High throughput flow cytometry system and method

ABSTRACT

The invention provides systems, compositions, kits and methods for automated processing of biological samples and analysis using a flow cytometer.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.14/667,388, filed Mar. 24, 2015, which is a continuation of U.S.application Ser. No. 12/606,869, filed Oct. 27, 2009, which claimspriority to U.S. Provisional Applications Nos. 61/108,803 filed Oct. 27,2008, 61/162,598 filed Mar. 23, 2009, and 61/181,211 filed May 26, 2009.Each of these applications is hereby expressly incorporated by referencein their entirety.

BACKGROUND OF THE INVENTION

Cell preparation for analysis using a flow cytometer is commonly usedfor monitoring progression of disease, monitoring therapy, and forscreening compounds that may be used to treat a disease.

Current techniques offer little in the way of automation for preparingsamples and analyzing them using flow cytometry. Current protocols forcell preparation are mostly performed by human operators. This meansthat sample throughput is limited to what a single person or team ofpeople can accomplish in a given amount of time.

Accordingly, there is a need for a process that has the advantage ofgenerating less labor intensive and more reproducible results. It isadvantageous to generate reliable data from highly controlled complexlaboratory automation for the study of cell signaling and for drugscreening either in the form of complete walk-away automation or withuser intervention at defined points. A highly controlled automatedprocess can increase throughput, reduce the potential for introducingvariability into the results, and ensure that meaningful results aregenerated.

SUMMARY OF THE INVENTION

The present invention relates to the processing of cells for analysis.More specifically, the present invention relates to a high throughputprocess and system for analyzing cells using a flow cytometer.

In accordance with one embodiment described herein, a system isdescribed that facilitates automated and high-throughput processing ofcell samples for flow cytometry. In some embodiments, the inventiondescribes a system that facilitates automated and high-throughputprocessing of cell samples for phosphoflow cytometry. In someembodiments, the invention describes a system that facilitates automatedand high-throughput processing of cell samples for analyzing receptorfunction (e.g. drug transporter) using flow cytometry. This systemincludes one or more robotic liquid handling instruments capable ofperforming sample transfer, reagent addition, mixing, aspiration,incubation, and centrifugation on a plurality of microplates orcartridges containing biological materials. One embodiment is comprisedof multiple automation systems that perform fewer functions and mayrequire some human intervention. One embodiment is a fully automatedsystem.

Particular embodiments are integrated systems for performing flowcytometry assays, acquisition, compilation, storage, and analysis ofdata. These systems may include one or more robotic liquid handlinginstruments with or without barcode scanners, operably linked to one ormore digital computers with software including an instruction set, aninput device, and a user interface to input plate data.

In a particular embodiment, automated and semi-automated instruments, aswell as peripheral equipment, exchange sample information and otherrelevant processing data with a laboratory integrated management system(LIMS). In a particular embodiment, applications and instruments maycommunicate with, or receive information from LIMS.

In a particular embodiment, the cytometer is part of the automatedplatform which may contain a liquid handler, automated centrifuge(V-spin), incubator, Hypercyte, cytometer controlled by a schedulingsoftware, and a pathway analysis tool at the back end. In a particularembodiment, the system be an automated approach to pathway profilingstarting from sample (ie, blood, marrow, purified cells, cell lines) andending with a full signaling pathway work-up. In a particularembodiment, the system may be an automated or semi-automated instrumentthat may be operably linked to a computational device equipped with userinput and output features.

In accordance with a particular embodiment, a method is described forautomatic retrieval of a plurality of microplates, as well as automaticsample transfer, reagent addition, mixing, aspiration, incubation, andcentrifugation of those microplates. The method may include the steps ofretrieving a selected microplate from the plate holder, performingspecified steps of the assay on that plate in a controlled and timedfashion, and then the final step of returning the completed microplateto the plate holder.

Applications of the present invention may include probing the variouspathways by analyzing responses to a host of stimuli. Applications mayalso include using the system in combination with a compound library toassess the inhibition or enhancement of various signaling nodes,assessing expression level and functionality of various drugtransporters, or probing the signaling differences among various cancercell lines (ie, NCI 60 cell lines). Examples of drug transportersinclude, but are not limited to, glycoprotein (MDR1), MDR-associatedprotein and breast cancer resistance protein. Particular embodiments maybe used to screen the supernatants from hybridomas in order to pick thebest antibody secreting cell clone. For instance, one could screen for aneutralizing or agonistic antibody to a growth factor receptor bylooking for the supernatant that best augments the signaling downstreamof the growth factor receptor. In another embodiment one could use theplatform to select the best cell clone secreting any protein that has abiological effect that could be measured by its downstream effect onsignaling.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a partially automated system that facilitateshigh-throughput processing of cell samples for phosphoflow cytometry.

FIG. 2 illustrates a partially automated system that facilitateshigh-throughput processing of cell samples for phosphoflow cytometry.

FIG. 3A illustrates a fully automated system that facilitateshigh-throughput processing of cell samples for phosphoflow cytometry.

FIG. 3B illustrates a fully automated system that facilitateshigh-throughput processing of cell samples for phosphoflow cytometry.

FIG. 4 illustrates an instrument for processing samples.

FIG. 5 illustrates an example method for sample preparation.

FIG. 6 illustrates an example apoptosis assays method.

FIG. 7 illustrates an example cell stimulation assays method.

FIG. 8 illustrates an example kinetic timepoint assay method.

FIG. 9 illustrates an example efflux assay method.

DETAILED DESCRIPTION OF THE INVENTION

High throughput flow cytometry system and method has many preferredembodiments and relies on many patents, applications and otherreferences for details known to those of the art. Therefore, when apatent, application, or other reference is cited or repeated below, itshould be understood that it is incorporated by reference in itsentirety for all purposes as well as for the proposition that isrecited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Particular embodiments of the high throughput flow cytometry system aredesigned to automate and improve the throughput of flow cytometrysystems, especially those that analyze cell signaling networks and cellreceptor function. Particular embodiments of the automated systemprovide a highly controlled platform to screen large numbers of samplesthat may then be analyzed using multiparametric flow cytometry.Particular embodiments of the automated system may be used in researchand development, diagnostic testing, clinical trials, testing celllines, drug screening and drug discovery applications. Particularembodiments of the automated system may be designed to reduce processingcost, reduce processing variability, or improve data integrity.

Particular embodiments comprise fully integrated, highly controlledautomated sample processing. In a particular embodiment, a robotic armmay operate around a variety of instruments that are involved in samplepreparation for the analysis. Those instruments may include a flowcytometer, a sample acquisition device (Hypercyt for example),microcentrifuge tubes holding cell samples, microplates holding cellsamples, centrifuges, reagent containers, temperature controlledincubators, thermal cyclers, temperature controlled refrigeration units,liquid handling devices, automated pipetting devices, automateddispensers, plate sealers, plate piercers, barcode scanners, plateholders and may be operated by a computer running the appropriatesoftware using the appropriate graphical user interface (GUI), which maybe integrated into a laboratory information management system (LIMS). Ina particular embodiment, the robotic arm may remove microplatescontaining cell samples from an incubator or plate holder, the plateswould be placed on a deck as a working surface to interact with otherinstruments and to accept the appropriate reagents. A liquid handling ormicropetting assembly may pull reagents from a plate and add them to thecells (or vice versa). Scheduling software may measure the appropriateincubation times and stop the reaction when necessary. The cycles may berepeated as required for the stimulation and preparation of the cellsfor flow cytometer analysis and the cells may be centrifuged and washedto prepare a pellet. The robotic arm may take the cell sample and passto a refrigeration unit for preservation or may subject it to immediateanalysis.

Particular embodiments comprise islands of automation requiring humanintervention to move samples from one process to the next. In aparticular embodiment a plurality of components are involved with samplepreparation for sample analysis. Those components may include a flowcytometer, a sample acquisition device (Hypercyt for example),microcentrifuge tubes holding cell samples, microplates holding cellsamples, centrifuges, reagent containers, temperature controlledincubators, thermal cyclers, temperature controlled refrigeration units,liquid handling devices, automated pipetting devices, automateddispensers, plate sealers, plate piercers, barcode scanners, plateholders and each instrument may be operated by computers running theappropriate software using the appropriate graphical user interface(GUI), which may be integrated into a laboratory information managementsystem (LIMS).

Background Information

Particular embodiments of the high throughput flow cytometry system andmethod incorporate information disclosed in other applications andtexts. The following patents and other publications are herebyincorporated by reference in their entireties: Haskell et al, CancerTreatment, 5^(th) Ed., W.B. Saunders and Co., 2001; Alberts et al., TheCell, 4^(th) Ed., Garland Science, 2002; Vogelstein and Kinzler, TheGenetic Basis of Human Cancer, 2d Ed., McGraw Hill, 2002; Michael,Biochemical Pathways, John Wiley and Sons, 1999; Weinberg, The Biologyof Cancer, 2007; Immunobiology, Janeway et al. 7^(th) Ed., Garland, andLeroith and Bondy, Growth Factors and Cytokines in Health and Disease, AMulti Volume Treatise, Volumes 1A and 1B, Growth Factors, 1996. Patentsand applications that are also incorporated by reference include U.S.Pat. Nos. 7,314,595, 7,368,084, 7,381,535 and 7,393,656 and U.S. patentapplication Ser. Nos. 10/193,462; 11/655,785; 11/655,789; 11/655,821;11/338,957, 61/048,886; 61/048,920; 61/048,657; 61/055,362; 61/079,766;61/085,789; 61/079,551; 61/079,537; 61/079,579; 61/087,555; 61/120,320;and 61/153,627. Relevant articles include High-content single-cell drugscreening with phosphospecific flow cytometry, Krutzik et al., NatureChemical Biology, 23 Dec. 2007; Irish et al., FLt3 ligand Y591duplication and Bcl-2 over expression are detected in acute myeloidleukemia cells with high levels of phosphorylated wild-type p53,Neoplasia, 2007, Irish et al. Mapping normal and cancer cell signalingnetworks: towards single-cell proteomics, Nature, Vol. 6 146-155, 2006;and Irish et al., Single cell profiling of potentiated phospho-proteinnetworks in cancer cells, Cell, Vol. 118, 1-20 Jul. 23, 2004; Schulz, K.R., et al., Single-cell phospho-protein analysis by flow cytometry, CurrProtoc Immunol, 2007, 78:8 8.17.1-20; Krutzik, P. O., et al., Coordinateanalysis of murine immune cell surface markers and intracellularphosphoproteins by flow cytometry, J Immunol. 2005 Aug. 15;175(4):2357-65; Krutzik, P. O., et al., Characterization of the murineimmunological signaling network with phosphospecific flow cytometry, JImmunol. 2005 Aug. 15; 175(4):2366-73; Shulz et al., Current Protocolsin Immunology 2007, 78:8.17.1-20; Stelzer et al. Use of MultiparameterFlow Cytometry and Immunophenotyping for the Diagnosis andClassification of Acute Myeloid Leukemia, Immunophenotyping, Wiley,2000; and Krutzik, P. O. and Nolan, G. P., Intracellular phospho-proteinstaining techniques for flow cytometry: monitoring single cell signalingevents, Cytometry A. 2003 October; 55(2):61-70; Hanahan D., Weinberg,The Hallmarks of Cancer, CELL, 2000 Jan. 7; 100(1) 57-70; Krutzik et al,High content single cell drug screening with phosphospecific flowcytometry, Nat Chem Biol. 2008 February; 4(2):132-42. Experimental andprocess protocols and other helpful information can be found athttp:/proteomices.stanford.edu. Articles and other references that maybe cited below are also incorporated by reference in their entiretiesfor all purposes. Some commercial reagents, protocols, software andinstruments that are useful in particular embodiments of the presentinvention are available at the Becton Dickinson Websitehttp://www.bdbiosciences.com/features/products/, and the Beckman Coulterwebsite, http://www.beckmancoulter.com/Default.asp?bhfv=7.

In addition to the references cited above, the practice of the presentinvention may employ, unless otherwise indicated, conventionaltechniques and descriptions of flow cytometry, organic chemistry,polymer technology, molecular biology (including recombinanttechniques), cell biology, cancer biology, biochemistry, and immunology,which are within the skill of the art. Such conventional techniques canbe found in articles, patents, commercial websites, as well as othersources as referenced above. Other conventional techniques can be shownin standard laboratory manuals such as those recited above and alsoincluding Genome Analysis: A Laboratory Manual Series (Vols. I-IV),Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCRPrimer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual(all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995)Biochemistry (4^(th) Ed.) Freeman, N.Y., Gait, “OligonucleotideSynthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox(2000), Lehninger, Principles of Biochemistry 3^(rd) Ed., W.H. FreemanPub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5^(th) Ed.,W.H. Freeman Pub., New York, N.Y., all of which are herein incorporatedin their entirety by reference for all purposes.

Use of Computers

Particular embodiments of high throughput flow cytometry system may beimplemented in a localized or distributed computing environment. Forexample, in a particular embodiment featuring a localized computingenvironment, a flow cytometry system may be operably linked to acomputational device equipped with user input and output features. In adistributed environment, the methods may be implemented on a singlecomputer, a computer with multiple processes or, alternatively, onmultiple computers. The computers may be linked, e.g., through a sharedbus, but more commonly, the computer(s) are nodes on a network. Thenetwork may be generalized or dedicated, at a local level or distributedover a wide geographic area. In particular embodiments, the computersmay be components of an intranet or an internet. See U.S. PatentApplication No. 61/048,657 and U.S. Pat. No. 7,314,595 which areincorporated by reference in its entirety for details of some computerhardware that may be useful in the present invention.

Particular embodiments also contemplate the use of a computer which mayoperate various instrumentation components, liquid handling ormicropipetting equipment, analysis instruments or analysis software. Thecomputer may be any type of computer platform such as a workstation, apersonal computer, a server, or any other present or future computer.The computer typically includes known components such as a processor, anoperating system, system memory, memory storage devices, andinput-output controllers, input-output devices, and display devices.Display devices may include display devices that provides visualinformation, this information typically may be logically and/orphysically organized as an array of pixels. A Graphical the userinterface (GUI) controller may also be included that may comprise any ofa variety of known or future software programs for providing graphicalinput and output interfaces such as for instance GUI's. For example,GUI's may provide one or more graphical representations to a the user,and also be enabled to process the user inputs via GUI's using means ofselection or input known to those of ordinary skill in the related art.

It will be understood by those of ordinary skill in the relevant artthat there are many possible configurations of the components of acomputer and that some components that may typically be included in acomputer are not shown, such as cache memory, a data backup unit, andmany other devices. The processor may be a commercially availableprocessor such as an Itanium® or Pentium® processor made by IntelCorporation, a SPARC® processor made by Sun Microsystems, an Athalon™ orOpteron™ processor made by AMD corporation, or it may be one of otherprocessors that are or will become available. Some embodiments of theprocessor may also include what are referred to as Multi-core processorsand/or be enabled to employ parallel processing technology in a singleor multi-core configuration. For example, a multi-core architecturetypically comprises two or more processor “execution cores”. In thepresent example each execution core may perform as an independentprocessor that enables parallel execution of multiple threads. Inaddition, those of ordinary skill in the related will appreciate thatthe processor may be configured in what is generally referred to as 32or 64 bit architectures, or other architectural configurations now knownor that may be developed in the future.

The processor executes an operating system, which may be, for example, aWindows®-type operating system (such as Windows® XP or Windows® Vista)from the Microsoft Corporation; the Mac OS X operating system from AppleComputer Corp. (such as 7.5 Mac OS X v10.4 “Tiger” or 7.6 Mac OS X v10.5“Leopard” operating systems); a Unix® or Linux-type operating systemavailable from many vendors or what is referred to as an open source;another or a future operating system; or some combination thereof. Theoperating system interfaces with firmware and hardware in a well-knownmanner, and facilitates processor in coordinating and executing thefunctions of various computer programs that may be written in a varietyof programming languages. The operating system, typically in cooperationwith the processor, coordinates and executes functions of the othercomponents of computer. The operating system also provides scheduling,input-output control, file and data management, memory management, andcommunication control and related services, all in accordance with knowntechniques.

The system memory may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, or other memorystorage device. Memory storage devices may be any of a variety of knownor future devices, including a compact disk drive, a tape drive, aremovable hard disk drive, USB or flash drive, or a diskette drive. Suchtypes of memory storage devices typically read from, and/or write to, aprogram storage medium (not shown) such as, respectively, a compactdisk, magnetic tape, removable hard disk, USB or flash drive, or floppydiskette. Any of these program storage media, or others now in use orthat may later be developed, may be considered a computer programproduct. As will be appreciated, these program storage media typicallystore a computer software program and/or data. Computer softwareapplications, also called computer control logic, typically are storedin system memory and/or the program storage device used in conjunctionwith memory storage device.

Input-output controllers could include any of a variety of known devicesfor accepting and processing information from a user, whether a human ora machine, whether local or remote. Such devices include, for example,modern cards, wireless cards, network interface cards, sound cards, orother types of controllers for any of a variety of known input devices.Output controllers of input-output controllers could include controllersfor any of a variety of known display devices for presenting informationto a user, whether a human or a machine, whether local or remote. In aparticular embodiment, the functional elements of computer maycommunicate with each other via system bus. Some of these communicationsmay be accomplished in alternative embodiments using network or othertypes of remote communications.

In a particular embodiment, the computer implemented methods and/orsystems may be operated locally on a computer, on a network serveraccessed through one or more terminals, or through one or more machinesaccessed through an intranet or internet. The method may also beimplemented through a computer readable medium storing a computerprogram implementing the method's functionality.

In particular embodiments, a computer application product may comprise acomputer-readable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by a processor, causes the processor to perform functionsdescribed herein. In particular embodiments, some functions may beprimarily implemented in hardware using a hardware state machine.Implementation of the hardware state machine so as to perform thefunctions described herein will be apparent to those skilled in therelevant arts.

As will be evident to those skilled in the relevant art, an instrumentcontrol and image processing application, such as for instance animplementation of instrument control and image processing applications,if implemented in software, may be loaded into and executed from systemmemory and/or memory storage device. All or portions of the instrumentcontrol and image processing applications may also reside in a read-onlymemory or similar device of memory storage device, such devices notrequiring that the instrument control and image processing applicationsfirst be loaded through input-output controllers. It will be understoodby those skilled in the relevant art that the instrument control andimage processing applications, or portions of it, may be loaded byprocessor in a known manner into system memory, or cache memory (notshown), or both, as advantageous for execution. Library files,calibration data, experiment data, and internet client data may bestored in system memory. For example, experiment data could include datarelated to one or more experiments or assays such as excitationwavelength ranges, emission wavelength ranges, extinction coefficientsand/or associated excitation power level values, or other valuesassociated with one or more fluorescent labels. Additionally, internetclient may include an application enabled to accesses a remote serviceon another computer using a network that may for instance comprise whatare generally referred to as “Web Browsers”. In the present example somecommonly employed web browsers include Microsoft® Internet Explorer 7with SPI available from Microsoft Corporation, Mozilla Firefox® 1.5 fromthe Mozilla Corporation, Safari 2.0 from Apple Computer Corp., or othertype of web browser currently known in the art or to be developed in thefuture. In particular embodiments, the internet client may include, orcould be an element of, specialized software applications enabled toaccess remote information via a network such as network.

The network may include one or more of the many various types ofnetworks well known to those of ordinary skill in the art. For example,the network may include a local or wide area network that employs whatis commonly referred to as a TCP/IP protocol suite to communicate, thatmay include a network comprising a worldwide system of interconnectedcomputer networks that is commonly referred to as the internet, or couldalso include various intranet architectures. Those of ordinary skill inthe related arts will also appreciate that some the users in networkedenvironments may prefer to employ what are generally referred to as“firewalls” (also sometimes referred to as Packet Filters, or BorderProtection Devices) to control information traffic to and from hardwareand/or software systems. For example, firewalls may comprise hardware orsoftware elements or some combination thereof and are typically designedto enforce security policies put in place by the users, such as forinstance network administrators, etc.

Instrument control and image processing applications may comprise any ofa variety of known or future image processing applications. Typically,particular embodiments of applications may be loaded into system memoryand/or memory storage device.

In particular embodiments, applications may be stored on a server orcomputer that may be accessed located locally or remotely andcommunicate with one or more additional servers and/or one or more othercomputers/workstations or instruments. In a particular embodiment,applications may communicate with, and receive instruction orinformation from, or control one or more elements or processes of one ormore servers, one or more workstations, and one or more instruments orsystem components.

In particular embodiments, automated and semi-automated instruments aswell as peripheral equipment exchange sample information with alaboratory integrated management system (LIMS) or laboratory informationsystem (LIS). In a particular embodiment, applications and componentsmay communicate with, or receive information from LIMS or LIS. LIMS orLIS is typically associated with a computer workstation containingsoftware applications, GUI, or instruments, and automates sampletracking from initial input into the system to data analysis. Forexample, see U.S. Patent Application No. 61/079,537.

Those skilled in the art will appreciate that applications may be storedfor execution on any compatible computer system, such as computer, orany compatible computer readable tangible medium. Embodiments ofapplications may advantageously provide what is referred to as a modularinterface for one or more computers or workstations and one or moreservers, as well as one or more instruments. The term “modular” as usedherein generally refers to elements that may be integrated to andinteract with a core element in order to provide a flexible, updateable,and customizable platform. For example, as will be described in greaterdetail below applications may comprise a “core” software element enabledto communicate and perform primary functions necessary for anyinstrument control and image processing application. Such primaryfunctionality may include communication over various networkarchitectures, or data processing functions such as processing rawintensity data into a .data file. In a particular embodiment, modularsoftware elements, such as for instance what may be referred to as aplug-in module, may be interfaced with the core software element toperform more specific or secondary functions, such as for instancefunctions that are specific to particular instruments. In particular,the specific or secondary functions may include functions customizablefor particular applications desired by the user. Further, integratedmodules and the core software element are considered to be a singlesoftware application, and referred to as applications.

Web Site Embodiment

A website or alternatively what is known in the art as “cloud computing”embodiment may be used where typically; a user (e.g., a scientist)executes a Web browser and is linked to a server computer executing aWeb server. In the present example some commonly employed web browsersinclude Microsoft® Internet Explorer 7 with SPI available from MicrosoftCorporation, Mozilla Firefox® 1.5 from the Mozilla Corporation, Safari2.0 from Apple Computer Corp., or other type of web browser currentlyknown in the art or to be developed in the future. The Web server may bea program such as IBM's HTTP Daemon or other WWW daemon (e.g.,LINUX-based forms of the program). The client computer may bebi-directionally coupled with the server computer over a line or via awireless system. In turn, the server computer may be bi-directionallycoupled with a website (server hosting the website) providing access tosoftware implementing the methods of this invention.

In particular embodiments, a user of a client connected to the Intranetor Internet may cause the client to request resources that are part ofthe web site(s) hosting the application(s) providing an implementationof the methods or systems of this invention. Server program(s) or APIsrunning on the server then process the request to return the specifiedresources (assuming they are currently available), e.g. a browserrunning an application that displays the selected visualizations andmetrics of an implementation described above. A standard namingconvention has been adopted, known as a Uniform Resource Locator(“URL”). This convention encompasses several types of location names,presently including subclasses such as Hypertext Transport Protocol(“http”), File Transport Protocol (“ftp”), gopher, and Wide AreaInformation Service (“WAIS”).

In a particular embodiment, when a resource is downloaded, it mayinclude the URLs of additional resources. Thus, the user of the clientmay easily learn of the existence of new resources that he or she hadnot specifically requested. One example of such an implementation wouldbe a user on a remote machine using a web browser to make procedurecalls, such as through an implementation of XML-RPC, discussed athttp://www.xmlrpc.com, or Simple Object Access Protocol (SOAP), to aserver application with an API for implementing the system or methods ofthe invention. The user through the web browser may be able to accessall system functionality through a remote location with a server runningthe applications implementing the methods or systems of the invention.

Methods of implementing Intranet and/or Intranet embodiments ofcomputational and/or data access processes are well known to those ofskill in the art and are documented, e.g., in ACM Press, pp. 383-392;ISO-ANSI, Working Draft, “Information Technology-Database Language SQL”,Jim Melton, Editor, International Organization for Standardization andAmerican National Standards Institute, July 1992; ISO Working Draft,“Database Language SQL-Part 2: Foundation (SQL/Foundation)”,CD9075-2:199.chi.SQL, Sep. 11, 1997; and Cluer et al. (1992) A GeneralFramework for the Optimization of Object-Oriented Queries, Proc SIGMODInternational Conference on Management of Data, San Diego, Calif., Jun.2-5, 1992, SIGMOD Record, vol. 21, Issue 2, June, 1992; Stonebraker, M.,Editor. Other resources are available, e.g., from Microsoft, IBM, Sunand other software development companies.

Integrated Systems

Integrated systems, e.g., for performing flow cytometry assays and dataanalysis, as well as for the compilation, storage and access ofdatabases, typically include a digital computer with software includingan instruction set as described herein, and, optionally, one or more ofhigh-throughput sample control software, image analysis software, otherdata interpretation software, a robotic control armature fortransferring solutions from a source to a destination (such as adetection device) operably linked to the digital computer, an inputdevice (e.g., a computer keyboard) for entering subject data to thedigital computer, or to control analysis operations or high throughputsample transfer by the robotic control armature. Optionally, theintegrated system may further comprise an image scanner for digitizinglabel signals from labeled assay components.

In particular embodiments, readily available computational hardwareresources using standard operating systems may be employed and modifiedaccording to the teachings provided herein, e.g., a PC running forexample, a Windows®-type operating system (such as Windows® XP orWindows® Vista) from the Microsoft Corporation; the Mac OS X operatingsystem from Apple Computer Corp. (such as 7.5 Mac OS X v10.4 “Tiger” or7.6 Mac OS X v10.5 “Leopard” operating systems); a Unix® or Linux-typeoperating system available from many vendors or what is referred to asan open source; another or a future operating system; or somecombination thereof for use in the integrated systems of the invention.Current art in software technology is adequate to allow implementationof the methods taught herein on a computer system. Thus, in particularembodiments, the present invention may comprise a set of logicinstructions (either software, or hardware encoded instructions) forperforming one or more of the methods as taught herein. For example,software for providing the described data, visualization, and/orstatistical analysis may be constructed by one of skill using a standardprogramming language such as Python, Visual Basic, Fortran, Basic, Java,C++, C#, or the like. Such software may also be constructed utilizing avariety of statistical programming languages, toolkits, or libraries.

In particular embodiments, various programming methods and algorithms,including genetic algorithms and neural networks, may be used to performaspects of the data collection, correlation, and storage functions, aswell as other desirable functions, as described herein. In addition,digital or analog systems such as digital or analog computer systems maycontrol a variety of other functions such as the display and/or controlof input and output files. Software for performing the visualizationmethods of the invention, such as programmed embodiments of thevisualization methods described above, are also included in the computersystems of the invention. Alternatively, programming elements forperforming such methods as principle component analysis (PCA) or leastsquares analysis may also be included in the digital system to identifyrelationships between data. Exemplary software for such methods isprovided by Partek, Inc., St. Peter, Mo; available athttp://www.partek.com. Optionally, the integrated systems of theinvention include an automated workstation.

In particular embodiments, automated and/or semi-automated methods forsolid and liquid phase high-throughput sample preparation and evaluationare available, and supported by commercially available devices. Forexample, robotic devices for preparation of nucleic acids from bacterialcolonies, e.g., to facilitate production and characterization of thelibraries of candidate genes include, for example, an automated colonypicker (e.g., the Q-bot, Genetix, U.K.) capable of identifying,sampling, and inoculating up to 10,000/4 hrs different clones into 96well microtiter dishes. Alternatively, or in addition, robotic systemsfor liquid handling are available from a variety of sources, e.g.,automated workstations like the automated synthesis apparatus developedby Takeda Chemical Industries, LTD. (Osaka, Japan) and many roboticsystems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; Orca, Beckman Coulter, Inc. (Fullerton, Calif.)) whichmimic the manual operations performed by a scientist. Additionally,incubators, refrigerators, freezers, plate sealers, reagent dispensers,and barcode scanners may be necessary to augment the robotic systems atthe appropriate processing steps. Any of the above devices are suitablefor use with the present invention, e.g., for high-throughput analysisof library components or subject samples. The nature and implementationof modifications to these devices (if any) so that they may operate asdiscussed herein will be apparent to persons skilled in the relevantart.

A variety of commercially available peripheral equipment, including,e.g., flow cytometers and related optical and fluorescent detectors, andthe like, and software are available for digitizing, storing andanalyzing a digitized video or digitized optical or other assay resultsusing a computer. Commercial Suppliers of flow cytometry instrumentationinclude Beckman Coulter, Inc. (Fullerton, Calif.), and Becton Dickinson(San Jose, Calif.), among many others.

In particular embodiments, applications may be capable of dataencryption/decryption functionality. For example, it may be desirable toencrypt data, files, information associated with GUI's or otherinformation that may be transferred over network to one or more remotecomputers or servers for data security and confidentiality purposes. Insome applications, the one-way encrypted data may be stored in one ormore public databases or repositories where even the curator of thedatabase or repository would be unable to associate the data with theuser or otherwise decrypt the information. The described encryptionfunctionality may also have utility in clinical trial applications whereit may be desirable to isolate one or more data elements from each otherfor the purpose of confidentiality and/or removal of experimentalbiases.

Particular embodiments provide one or more interactive graphical userinterfaces that allow the user to make selections based upon informationpresented in an embodiment of GUI. Those of ordinary skill willrecognize that embodiments of GUI may be coded in various languageformats such as an HTML, XHTML, XML, javascript, Jscript, or otherlanguage known to those of ordinary skill in the art used for thecreation or enhancement of “Web Pages” viewable and compatible withinternet client, or other languages known or developed in the future foruse in the creation of computer GUIs. For example, the internet clientmay include various internet browsers such as Microsoft InternetExplorer, Mozilla Firefox, Apple Safari, or other browsers known in theart. Applications of GUI's viewable via one or more browsers may allowthe user complete remote access to data, management, and registrationfunctions without any other specialized software elements. Theapplications may provide one or more implementations of interactiveGUI's that allow the user to select from a variety of options includingdata selection, experiment parameters, customization features, desiredvisualizations, metrics and pre-filters.

In particular embodiments, the applications may be capable of running onoperating systems in a non-English format, where applications may acceptinput from the user in various non-English language formats such asChinese, French, Spanish, etc., and output information to the user inthe same or other desired language output. For example, applications maypresent information to the user in various implementations of GUI in alanguage output desired by the user, and similarly receive input fromthe user in the desired language. In the present example, theapplications are internationalized such that it is capable ofinterpreting the input from the user in the desired language where theinput is acceptable input with respect to the functions and capabilitiesof the applications.

In particular embodiments, it may be desirable to consolidate elementsof data or metadata related to an experiment, the user, or somecombination thereof, to a single file that is not duplicated whereduplication may sometimes be a source of error. The term “metadata” asused herein generally refers to data about data. In particularembodiments, it may also be desirable in some embodiments to restrict orprohibit the ability to overwrite data in the file. Preferentially, newinformation may be appended to the file rather than deleting oroverwriting information, providing the benefit of traceability and dataintegrity (e.g. as may be required by some regulatory agencies).

The invention also may be embodied in whole or in part within thecircuitry of an application specific integrated circuit (ASIC) or aprogrammable logic device (PLD). In such a case, the invention may beembodied in a computer understandable descriptor language, which may beused to create an ASIC, or PLD that operates as herein described.

In a particular embodiment, a system to provide notice to the operatorsof the cytometer lab that the samples are in transit may be conductedover the internet. U.S. Patent Publication No. 20050009078 discusses theuse of internet ordering systems useful in the present invention.

Certain functional elements, such as files and data structures, may bedescribed in the illustrated embodiments as located in system memory ofa particular computer. In particular embodiments, however, they may belocated on, or distributed across, computer systems or other platformsthat are co-located and/or remote from each other. For example, any oneor more of data files or data structures described as co-located on and“local” to a server or other computer may be located in a computersystem or systems remote from the server.

In addition, it will be understood by those skilled in the relevant artthat control and data flows between and among functional elements andvarious data structures may vary in many ways from the control and dataflows described above or in documents incorporated by reference herein.More particularly, in particular embodiments, intermediary functionalelements may direct control or data flows, and the functions of variouselements may be combined, divided, or otherwise rearranged to allowparallel processing or for other reasons. In particular embodiments,intermediate data structures or files may be used and various describeddata structures or files may be combined or otherwise arranged. Numerousother embodiments, and modifications thereof, are contemplated asfalling within the scope of the present invention as defined by appendedclaims and equivalents thereto.

Methods

Particular embodiments of the automation system perform various methodsof cell preparation for multiparametric analysis on a flow cytometer. Itis understood by the skilled artisan that the steps of the methodsprovided herein may vary in order. It is also understood, however, thatwhile various options (of reagents, handling properties selected, ororder of steps) are provided herein, the options are also each providedindividually, and may each be individually segregated from the otheroptions provided herein. Moreover, steps that are known in the art thatmay increase the sensitivity of the assay are intended to be within thescope of this invention. For example, there may be additional washingsteps, blocking steps, etc. It is understood by the skilled artisan thatsteps of the methods provided herein may vary based on application, suchthat some steps may be added or deleted depending on the application.

FIG. 1 illustrates a high throughput system and method for highthroughput flow cytometry 100. In step 101, cell samples may be preparedfor processing. In step 103, cells are contacted with one or moremodulators in to stimulate the cells, then the cells are fixed with abuffer containing reagent. Then the cells may be permeabilized in step105 to allow reagent access across the cell membrane. In step 107,additional reagents are contacted with the cells to “stain” particularproteins within the cell. Thereafter, the cells are analyzed on a flowcytometer in step 109 to detect the presence or absence of the stains.See U.S. Pat. Nos. 7,381,535 and 7,393,656 or application Ser. Nos.10/193,462; 11/655,785; 11/655,789; 11/655,821; and 11/338,957 which areall incorporated by reference in their entireties. See also 61/048,886;61/048,920; 61/048,657; 61/079,766, and 61/085,789 which are also herebyincorporated. As illustrated in FIG. 5-FIG. 9, high through system andmethod for high throughput flow cytometry 100 may be utilized with avariety of applications that all result in analysis of cells on a flowcytometer, as illustrated in step 109.

The instant invention also makes use of cells that have been“potentiated.” In contrast to “activation,” a “potentiated” state refersthe state of a cell after exposure to a modulator which then may beactivated as the case may be. As described in detail below, modulatorsexert their effect on signaling cascades by directly or indirectlyimpacting the ability of an activatible protein to switch betweenactivation isoforms.

Sample Acquisition

Many of the methods and applications described herein require samples ofcells on which to perform various analyses. Cells may be acquired fromvariety of sources using a variety of techniques. Regardless of thecollection technique used, it is important that there are a sufficientnumber of cells for the flow analysis. In particular embodiments, cellsmay be acquired from an individual, by a blood draw, a marrow draw, or atissue extraction. In particular embodiments, cells may be acquired by avariety of other techniques and may include sources, such as bonemarrow, solid tumors, ascites, washes and the like. In a particularembodiment, tissue may be taken from an individual using a surgicalprocedure. Surgical procedures to acquire tissue are well known in theart, as are subsequent processing of the tissue. Tissue may be fixed orunfixed, fresh or frozen, whole or disaggregated. Disaggregation oftissue may occur either mechanically or enzymatically. In particularembodiments, cells may be cultured. The cultured cells may be developedcell lines or patient derived cell lines. Procedures for cell cultureare commonly known in the art.

In particular embodiments, once cell samples have been collected theymay be stored for later usage, processed and stored for later usage,processed and used immediately, or simply used immediately. Inparticular embodiments, processing may include various methods oftreatment, isolation, purification, filtration, or concentration.Particular embodiments of high throughput flow cytometry system andmethod may utilize fresh or cryopreserved samples of blood, bone marrow,peripheral blood, tissue, or cell cultures. Particular embodiments mayutilized samples that are activated or unactivated, fixed or unfixed,lyophilized or in suspension. In a particular embodiment, processing maybe performed by an automated component, such as the micropipettingassembly illustrated in FIG. 4. In a particular embodiment, aninstrument may access the processing method by a computer readabletangible medium storing a computer program that contains processinginstructions for various types of cell samples.

In particular embodiments, preparation of the cell samples may involvemultiple steps. These steps may require multiple reagents at varioustimes in the process and it is preferable to add the reagents soon afterthe blood drawing. Preferably, the reagents are added within 0, 3, 5,10, 15, 20, 30, 45, or 50 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,15, 24, 30, or 36 hours after collection. In one embodiment, the cellsare collected directly by venous puncture and treated with the reagents.See U.S. Patent Application No. 61/048,657.

In particular embodiments, blood may be treated without processing(whole blood) or may be processed in some fashion. For example, subsetsof cells within the blood may be filtered and removed for further useand analysis using standard collection or filtering devices. Inparticular embodiments, samples may be whole blood, cell suspensions,cells in a buffy coat sample, or fixed to a solid substrate, such as abead or plate. Some cell concentration devices or techniques may beemployed by insertion between the blood collection tube and thechambers. In a particular embodiment, a filtering device that separatescells may be placed in a fluid line between the blood collection tubeand another component of the system. Such devices may include micro cardor lab-on-α-chip devices that separate specific subsets of cells fromthe whole blood. In a particular embodiment, whole blood may also beapplied to filters that are engineered to contain pore sizes that selectfor the desired cell type or class. For example, cells may be filteredout or diluted, whole blood following the lysis of red blood cells byusing filters with pore sizes between 5 to 10 μm, as disclosed in U.S.patent application Ser. No. 09/790,673. In a particular embodiment, alarger volume of blood may be necessary where the concentration of cellswas low and filters may be used to concentrate the cell sample. See U.S.patent application Ser. No. 09/790,630.

Alternately, after collection of the cells, particular embodiments mayplace cell samples into chambers for processing through a fluidconnection between the tube and the chambers See U.S. Patent ApplicationNo. 61/048,657. The fluid connection may be any one of a manner ofconduits (such as commercial lab tubing) of any appropriate size andshape. Tubing is commercially available through a variety of medical andresearch suppliers. See the commercially available equipment fromCorning, Becton-Dickinson, Sastedt, and Tygon.

Sample Processing

High throughput flow cytometry system and method is designed tofacilitate automated and high throughput processing of cell samplesusing phosphoflow cytometry techniques. High throughput flow cytometrysystem may be a flexible instrument-based system where one or moreinstruments may interact with each other. Furthermore, particularembodiments may integrate improved procedures to allow more samples tobe analyzed in fewer steps or to provide additional information to theuser. Particular embodiments may comprise several elements, such as oneor more instruments, stations, or computers. Particular embodiments maybe carried out by fewer elements or a single element. Additionally, anyor all of the steps outlined herein may be automated. In particularembodiments, high throughput flow cytometry system may be partially orcompletely automated.

FIG. 1 illustrates a particular embodiment of a system and method forhigh throughput flow cytometry 100. In FIG. 1, system and method forhigh throughput flow cytometry 100 comprises islands of automation 101,103, 105, 107, 109. In particular embodiments, any functional element ofFIG. 1 may perform fewer or different operations than those describedwith respect to the illustrated embodiment. Also, various functionalelements shown as distinct for purposes of illustration may beincorporated within other functional elements in a particularembodiment. In particular embodiments, the sequence of functions orportions of functions may be altered.

FIG. 2 illustrates a particular embodiment of a system and method forhigh throughput flow cytometry 200. In FIG. 2, system and method forhigh throughput flow cytometry 200 comprises islands of automation 201,203, and 205. In particular embodiments, any functional element of FIG.2 may perform fewer or different operations than those described withrespect to the illustrated embodiment. Also, various functional elementsshown as distinct for purposes of illustration may be incorporatedwithin other functional elements in a particular embodiment. Inparticular embodiments, the sequence of functions or portions offunctions may be altered.

FIG. 3A and FIG. 3B illustrate particular embodiments of a fullyautomated high throughput flow cytometry system 300 where theinstrumentation is within a single platform or footprint. As will beappreciated by those in the art, there are a wide variety of componentswhich may be used in association with the system. Components include,but are not limited to, fixed or mobile plate or reagent stages 301 withmicrofluidic systems; plate handlers 303 for the positioning ofmicroplates that may have automated lid or cap handlers to remove andreplace lids; one or more robotic arms 305 that may be centrallylocated, as illustrated in FIG. 3A, or proximately located to othercomponents, as illustrated in FIG. 3B; flow cytometer 307; automatedcentrifuges 309; heated, cooled, or ambient reagent racks or stackingtowers 311. Particular embodiments may have one or more of thecomponents previously mentioned and one or more computer systemsoperatively linked to one or more of those components. Particularembodiments may reduce in size over time as instrumentation decreases insize or systems may be simplified as processes become more efficient.Particular embodiments may include an automated approach to inventorymanagement in addition to pathway profiling starting from sampleacquisition and ending with a full signaling pathway work-up.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting and dispensing to perform all steps of screening applications.This includes liquid, particle, cell, and organism manipulations such asaspirating, dispensing, mixing, diluting, washing, accurate volumetrictransfers; retrieving and discarding pipet tips; and repetitivepipetting of identical volumes for multiple deliveries from a singlesample aspiration. These manipulations are cross-contamination-freeliquid, particle, cell, and organism transfers. This system performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and has high-capacity operation. In particular embodiments,robotic or microfluidic systems may manipulate any number of differentreagents, including modulators, buffers, fixatives, stains,permeabilizing reagents, reagent cocktails, samples, wash solutions,assay components such as label probes, etc.

Particular embodiments of high throughput flow cytometry system mayinclude one or more liquid handling components. The liquid handlingsystems themselves may include robotic systems comprising micropipettingassemblies and any number of other components, such as the componentsillustrated in FIG. 3A and FIG. 3B. Example instruments that may beuseful include, but are not limited to, the Sciclone series i1000 orALH3000 and its Autostacker, or the Zepher from Caliper, the Biomekseries, including the NX^(P), NX, FX and the FX^(P) from BeckmanCoulter, including the Bar Code Reading ALP, Stacker Carousel platehotel, Cytomat conveyor ALP, Automated Tube Bar Code Reader (oncemicroplates are formatted), or Handheld Bar Code Reader, the Velocity 11(Agilent) series, including the BioCel, BenchCel, Bravo, and Vprep, theTecan EVO liquid handling robots, the Platemate, Hyrdra, or Wellmateliquid handlers and dispensers from Matrix, or the Hamilton Roboticsliquid handling workstations such as the STAR or STARlet Lines, or theHyperCyt Autosampler by Intellicyt or other such autosamplers. All ofthe above instruments are shown on the websites for the relevantcompanies.

Micropippetting assemblies are well known in the art. See U.S. Pat. No.6,374,683. FIG. 4 illustrates an instrument 400 with a micropipettingassembly 401, a user interface 403, a mobile plate or reagent stage 405.Instrument 400 may be a stand-alone instrument or operably linked to acomputer system that contains software for instrument control andoperation.

The micropipetting assembly 401 may utilize syringe, positivedisplacement, air displacement, peristaltic, or other dispensingtechnology. The micropipetting assembly 401 may use fixed or disposabletips that may be washable in a wash station. The micropipetting assembly401 may be movable in at least two dimensions so that reagents orsamples may be transferred from a container to a microplate well, from amicroplate well to an alternate container, or between microplate wells.Particular embodiments of micropipetting assembly 401 may transferliquid reagents or samples to and from microplate wells and reagenttroughs. In a particular embodiment, micropipetting assembly maycomprise a plurality of independently controlled micropipetters. In aparticular embodiment, each micropipettor may be controlled separatelyfor aspiration and dispense volumes, for the precise microplate wellsthat it will aspirate or dispense from, and for timing of suchaspiration and dispenses.

User interface 403 allows an operator to manually interact with theinstrument to create or execute a program to control the operation ofthe instrument. Mobile plate or reagent stage 405 allows micropipettingassembly 401 to interface with one or more plates or other vessels thatmay contain reagents or sample. In a particular embodiment, mobile plateor reagent stage 405 may include a modular platform that may accommodateheated, cooled, or ambient deck positions, a variable speed orbitalshaker, positions for source and destination vessels, or sample andreagent reservoirs, pipette tips, and an active wash station. In aparticular embodiment, mobile plate or reagent stage may be accessibleby other components, such as a plate handler like the plate handler 305illustrated in FIG. 3.

In particular embodiments, reagents may be delivered by a dispensercapable of aliquotting fluids to individual wells along an X-Y-Z axis.See U.S. Pat. No. 6,121,048. In particular embodiments, the dispensermay utilize a piezoelectric pump, pipettes, micropipettes,electrophoretic pumps, or mechanisms adapted from ink-jet printingtechnology to dispense precise amounts of fluids. In a particularembodiment, reagents may be delivered to precise locations within aplate or cartridge. In a particular embodiment, specified reactants maybe delivered to certain wells which may be identified by encodedinformation, such as a bar code, RFID, or magnetic coding, controlled bya processor such as a computer.

Particular embodiments of high throughput flow cytometry system mayinclude thermoregulating components. Thermoregulating components may beused to stabilize the temperature of any number of samples or reagents.Thermoregulating components may include, but are not limited to,incubators, refrigerators, freezers, heat exchangers, such astemperature controlled blocks, resistive elements, thermoelectricmodules, or conductive elements. Thermoregulating components mayregulate temperatures ranging from −80° C. to 100° C. In particularembodiments, the thermoregulating component may include a temperaturedetection device, such as a thermocouple, which provides signalscorresponding to temperature readings to another component or acomputer. When a controller receives the signals corresponding to thetemperature readings it may adjust the power output in order to maintainthe selected temperature. In particular embodiments, thermoregulatingcomponents may be placed in close proximity to the other components forease of access. In a particular embodiment, robotic elements, such asrobotic arm 305, illustrated in FIG. 3A and FIG. 3B, may be used totransport microplates from the thermoregulating components to any othercomponent. In a system as illustrated in FIG. 1, microplates may betransferred to and from thermoregulating components by hand from oneautomated component to the next.

Optionally, a temperature control mechanism such as a heater, a cooler,or a combination thereof may be disposed next to the cartridge. Thetemperature control mechanism may be any suitable thermally controlledelement such as. The temperature control mechanism transfers heat orcold via conduction to the cartridge, which transfers heat or cold tofluid in the chamber. Alternatively, the temperature control mechanismsinks heat away from, for example, fluid in the chamber. The temperaturecontrol mechanism maintains a selected temperature in the chamber. Thetemperature control mechanism also

Particular embodiments of high throughput flow cytometry system mayutilize microtiter type plates. The plate may be conventional andcommercially available, or it may be a custom design. The number ofwells may be 96, 384, 1536 or other standard sizes. The volume may be asstated above, at least 1, 2, 3, 4, 5, 6, or 7 or more milliliters.Microtiter plates may be obtained from commercial suppliers such asthose listed above. In a particular embodiment, the microtiter plate mayhave predeposited reagents.

Particular embodiments of high throughput flow cytometry system mayinclude chemically derivatized particles, plates, cartridges, tubes,magnetic particles, or other solid phase matrix with specificity to theassay components are used. The binding surfaces of microplates, tubes orany solid phase matrices may include non-polar surfaces, highly polarsurfaces, modified dextran coating to promote covalent binding, antibodycoating, affinity media to bind fusion proteins or peptides,surface-fixed proteins such as recombinant protein A or G, nucleotideresins or coatings, and other affinity matrix are useful in thisinvention.

In a particular embodiment, plates or tubes may have preloaded reagents.In a particular embodiment, the plates may have separate wells orcompartments, and each well or compartment may be capable of containinga separate reagent. These reagents may be shown in the pending U.S.applications referred to above, such as U.S. Patent Application No.61/048,657 or 61/108,803.

Particular embodiments of high throughput flow cytometry system mayinclude platforms for multi-well plates, multi-tubes, holders,cartridges, minitubes, deep-well plates, microfuge tubes, cryovials,square well plates, filters, chips, beads, and other solid-phasematrices or platforms with various volumes are accommodated on anupgradable modular platform for additional processing. Particularembodiments may include a variable speed orbital shaker, andmulti-position work decks for source samples, source and destinationassay plates, sample and reagent reservoirs, pipette tips, and an activewash station.

Particular embodiments of the system may use components of an automatedhandling apparatus similar to the simplified schematic perspectivedrawing in FIG. 7 of U.S. Pat. No. 7,314,595. Example components includea microplate carousel optionally situated within environmental controlchamber. In some embodiments, one or more environmental control chambersmay also be provided for other components. Temperatures for the chambersmay range from a low of −80° C., −20° C., or 4° C. to highertemperatures of 30° C., 60° C., 95° C. or 100° C. In particularembodiments, the carousel and chamber may be structurally connected toother components by a frame. In a particular embodiment, a microplaterobot may selectively move a microplate stored in slots of carousel to amicroplate retainer by employing aspects of sensors and motors/actuatorsunder control of executables. In a particular embodiment, an operatormay manually place or remove the microplates from the chambers.

Particular embodiments of the system may include components, such as thecomponents illustrated in FIGS. 11-12 of U.S. Pat. No. 7,314,595. Forexample, FIG. 11 illustrates a robot that contains mechanisms forholding a microplate, removing its lid, and identifying the microplateby reading a one- or two-dimensional barcode or employing any of variousother machine-readable indicators (e.g., signal transmitter ortransducer, or other device, any one or more of which are referred tofor convenience herein simply with reference to “barcode”) known tothose of ordinary skill in the relevant art. In a particular embodimentthe robot may also return a microplate to the carousel after it has beenused, although microplates that have been processed may also be storedelsewhere. In a particular embodiment, the robot may not need to removethe lid prior to scanning the barcode or it may return it to analternate position within the instrument other than the carousel orplate stacker. In a particular embodiment, an operator may manually scanthe microplate or barcodes using a handheld barcode scanner.

Particular embodiments of the system may include microplate retainers,such as those illustrated in FIGS. 15A-C and 16A-C of U.S. Pat. No.7,314,595. In a particular embodiment, the microplate retainer mayutilize fiducial features for registering bottom surfaces of themicroplates to a retainer. (See also aspects of the use of referencemarks and other features and techniques as described in U.S. Pat. No.6,121,048, hereby incorporated herein by reference in its entirety forall purposes.)

In particular embodiments, once a microplate is secured in a retainer,sampling equipment may be lowered into selected or predeterminedmicroplate wells to add or remove reagents, cells or other reactants. Inparticular embodiments, the equipment may be entirely submerged in thefluid material in the microplate well. In particular embodiments, theequipment may either be kept stationery while the microplate is moved tothe equipment or move to where the microplate is situated. The fluidvolume in the equipment may be 0.001, 0.005, 0.01, 0.1, 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more microliters, but usually not more than 10,7.5, 5.0, 2.5 or 1.0 milliliters.

In particular embodiments of the system, flexible hardware and softwaremay allow instruments to be adaptable for multiple applications. In aparticular embodiment, the software program modules may allow creation,modification, and running of one or more methods. In a particularembodiment, the system diagnostic modules may allow for instrumentalignment, correct connections, and motor operations. Additionally,customized tools, labware, and liquid, particle, cell and organismtransfer patterns may allow different applications to be performed. In aparticular embodiment, the database may allow method and parameterstorage, as well as sample tracking or additional system controls. In aparticular embodiment, robotic and computer interfaces may allowcommunication between one or more instruments and LIMS or LIS. U.S.Patent Application Nos. 61/079,551 and 61/153,627 illustrate exampleinterfaces that are capable of storing experimental designs, platelayout, and inventory management, and are hereby incorporated byreference in their entirety. In a particular embodiment, the method maybe implemented through one or more instruments accessed by a computerreadable tangible medium storing a computer program implementing themethod's functionality.

Particular embodiments of high throughput flow cytometry system andmethod may include a system for automated processing in small volumesusing a sample processing cartridge. Typical reaction volumes in asample processing cartridge may be 1 μl to 100 ml. Particularembodiments are capable of distributing specified and accurate liquidvolumes to the selected chambers. Appropriate electronic and manualvalve devices may be used to ensure that liquid is placed in the correctchambers. For example, the micropipetting assemblies recited above maybe used to measure liquid volumes. Valves may include standard physicaldevices that are present on laboratory equipment that rotate with manualstopcocks or electronic valves, or may include the types of valves orliquid shunting devices or methods that are present with lab-on-α-chiptype devices where liquid volumes may be transferred to selectedchambers using positive or negative pressures or other valving. See forexample U.S. Pat. No. 6,830,936 and U.S. Publication Nos. 2007157973;20060258019; 20020079008; 20060134599; and 20020125139. See also Seiler,K. et al., “Electroosmotic Pumping and Valveless Control of Fluid FlowWithin a Manifold of Capillaries on a Glass Chip,” Anal. Chem.66:3485-3491 (1994); Cheng X, Irimia, et. al., Practical label-free CD4+T cell counting of HIV-infected subjects: A Microchip approach. Lab on aChip 2007; 10:1039; Demirci U, Toner M., Direct etch method formicrofluidic channel and nanoheight post-fabrication by picoliterdroplets, Applied Physics Letters 2006; 88 (5), 053117; and Irimia D,Geba D, Toner M., Universal microfluidic gradient generator, AnalyticalChemistry 2006; 78: 3472-3477.

In particular embodiments, if the cells of interest were concentrated,then smaller volume cartridges may be used, such as the lab-on-α-chipdevices, referred to above. For cartridges that are used in thesedevices a typical cartridge may be about 0.5 to 3″ wide, 0.5 to 4″ long,and 0.1 to 1″ high or more preferably 1 to 2″ wide, 1 to 3″ long, and0.5 to 1″ high. Preferably, the cartridge is of sufficient size toaccommodate identification labels, RFIDs, or bar codes in addition tothe chambers. Cartridges in which the chambers have a volume of between2 and 10 mls, may be between 2 to 10″ wide, 2 to 12″ long, and 1 to 3″high, or more preferably 5 and 9″ wide, 3 and 9″ long, and 1 to 2″ high.

In particular embodiments, the chamber of a cartridge may have anyconceivable size, shape, or orientation. Preferably, the chamber has avolume sufficient to allow the cells to contact one or more reagents,such as a modulator, and then be mixed with other reagents, like afixative, permeabilizing reagent or a stain. In a particular embodiment,the chamber may be at least 0.2″ wide, 0.2″ long, and 0.2″ deep. Forsmall volume chambers the volume may be in the microliter scale, such asthe volumes described above.

In particular embodiments, the cartridge may be open on two or more endsor access points. In a particular embodiment, the cell sample may beinserted through an inlet, port or opening. In a particular embodiment,there may be only one opening to insert the cell sample if theappropriate reagents are pre-deposited in the chambers awaiting cells.In a particular embodiment, the cartridge may be constructed to be ableto open it to insert the sample and then to close it to incubate orotherwise mix the sample plus the reagents. In a particular embodiment,the cartridge may have a separate access point to add the reagents tothe cell sample after the sample has been inserted into the chamber. Thereagents may be added individually or in one operation.

In particular embodiments, a separate device, such as an automateddispenser may add reagents to the cartridge. In particular embodiments,the automated dispenser may be attached to the cartridge through acassette. The use of a cassette having areas that align to the chambersof the cartridge may further enable high-throughput processing of thesamples in lieu of the slower process of individual addition. Inparticular embodiments, alignment marks and structures may be added tofacilitate an appropriate match between the cartridge and devices withthe reagents. Alternatively, the reagents may be added to the chambersin the cartridge through the manifold opening once the manifold isremoved.

In particular embodiments, selected fluids may be introduced into andout of the chamber via inlets. In particular embodiments, the inlets arelocated at opposite ends of the chamber. Having inlets located atopposite ends of the chamber may improve fluid circulation andregulation of bubble formation in the chamber for mixing. In aparticular embodiment, the bubbles may be used to agitate the fluid,thus increasing the contact between the one or more modulators and cellsamples. Other methods to mix the sample are known in the art. In aparticular embodiment, inlets are located at the top and bottom end ofthe chamber at opposite corners. Locating the inlet at the highest andlowest positions in the chamber may facilitate the removal of bubblesfrom the chamber if desired. In particular embodiments, internalstructure may also be used in the chamber to facilitate mixing. Suchstructure may be pegs, posts, or other physical structure that maydisrupt the fluid flow to enhance mixing.

In particular embodiments, the cartridge may contain chambers having aninlet for the cell sample and a separate inlet for the reagents. In aparticular embodiment, the reagents may be added by attaching astructure with or without its own chamber, which may align the reagentswith the chambers for contact with the cells. In a particularembodiment, the structure may also have a connection to add furtherreagents according to a particular timed schedule as shown in the patentapplications identified above.

In particular embodiments, once the first reagent is added, the cellsand the fluid in the chamber may be subject to agitation to improvecontact of the all of the reagents with the cells. See U.S. Pat. No.6,399,365 for examples of agitation systems. The agitation may involveexternal shaking or internal fluid circulation. Ports or inlet may beused to add further reagents. In particular embodiments, during any ofthese procedures, the manifold may be removed and the chamber openingsmay be covered with a seal to prevent leakage and evaporation. The cover(or any other seal) may be attached via clips, clamps, screws,adhesives, and other fasteners.

In particular embodiments, the cartridge may include a seal at anyopening of the chamber. In a particular embodiment, the seal may be aseptum composed of rubber, teflon/rubber laminate, or other sealingmaterial. The septum may be of the type commonly used to seal and resealvessels when a needle is inserted into the septum for addition/removalof fluids. The septums, when seated in the depressions, extend slightlyabove surface, which in some embodiments is about 0.01″ to 0.05″.

It should be noted that the even distribution of fluid flow through thechamber prevents dead zones from occurring in the chamber. For example,the even distribution of fluid through the chamber substantiallyprevents fluid from becoming substantially quiescent at certainlocations. It should also be noted that any of the components mentionedabove may be used in conjunction with the cartridge.

Timing

In particular embodiments, the system may control the timings for samplehandling, reagent addition, and incubation. For any given sample beingprocessed, there may be appropriate timings for sample handling, reagentaddition, and incubation, which may ensure data integrity and ease dataanalysis. In particular embodiments, timings for sample handling,reagent addition, and incubation may either be a very specific set time,for example, a fixative may need to incubate for exactly 10 minutes, ora range of acceptable times, for example, a stain may incubate a fewhours to overnight, producing acceptable results. In a particularembodiment, the system may treat each sample the same in terms oftimings at each step of the automated process for sample handling,reagent addition, and incubation.

Particular embodiments of the system may control timing in a variety ofways. In a particular embodiment, where the system is fully automated,the timing may be controlled by software, instruments, or otherauxiliary systems. In a particular embodiment, the system may assureproper timing of stimulation, incubation, and fixation by using a liquidhandling instrument to dispense a modulator and then a fixative into asample while controlling the timing of all other processes as well, suchas movement of the sample to shakers, incubators, hotels, or alternatepositions. In a particular embodiment, where the system is not fullyautomated, the system may utilize human intervention, such asoperator-implemented controls or batch processing to regulate thetiming. In a particular embodiment of the system, each batch willproceed through the process in the same controlled manner such that eachbatch will be treated the same at each step of the sample preparationprocess within the system. The batch size may be determined byinstrument limitations, limitations on sample handling, reagentaddition, or incubation times, as well as overall processing time persample or any number of other limitations imposed by the process itselfto maintain data integrity.

Particular embodiments of the system may incorporate an additionalmethod of handling appropriate timing with respect to reagent addition,such as a mechanism for timed release of reagents. Methods for timedrelease are known in the art and include physical structure or chemicalformulations that are designed to release a reagent at a particular timeafter an event like adding the sample. In a particular embodiment,physical devices may include an enclosure like an ampoule that may bebroken to release a reagent. In a particular embodiment, other physicaldevices may include structure having a porous nature that will release areagent over time, such as a porous membrane. Examples of chemicalformulations that allow the timed release of reagents include, but arenot limited to micelles, liposomes, cleavable linkers attached to thereagent and/or another molecule, and the like. See U.S. Pat. Nos.6,004,572, 5,079,005, and 5,023,080.

Particular embodiments of the system may use cartridges to prepare thesamples. In a particular embodiment, an instrument to process thesamples in the cartridge may control the timings for sample handling,reagent addition and incubation. In a particular embodiment, thereagents may be added to the cartridge chambers prior to the addition ofthe cell sample. This embodiment allows the cartridge to be preloadedand may make the process simpler for the user while still ensuringprocessing uniformity. In a particular embodiment, the reagents may beadded after the cell samples are added to the cartridge chambers. Thisembodiment may require manual addition of reagents and some localprocessing by the operator or an instrument or mechanism that wouldautomatically add the reagents to all chambers accurately andefficiently. Particular embodiments of the system may leave thecartridge attached or remove the cartridge and seal it from theenvironment.

System Controls

System controls are commonly used in the art to ease troubleshooting,maintain or improve throughput, and ensure end result data integrity.Particular embodiments of the automation system may have controls atvarious points within the system. Various system controls may beenvironmental controls, operator controls, reagent controls, instrumentcontrols, process quality control checkpoints, and protocol controls.

In a particular embodiment, a system control for managing and monitoringthe automation system may itself be a system for experimental design,layout, and inventory management. See U.S. Patent Application No.61/079,551, which is hereby fully incorporated by reference in itsentirety.

Particular embodiments of the automation system may have system controlsthat interface with the automation system by way of a laboratoryintegrated management system (LIMS) or a laboratory information system(LIS). Particular embodiments of a LIMS or LIS may be associated with acomputer workstation that may contain software applications, GUI, orinstruments, and may automate sample tracking from wherever the sampleis initially input into the automation system through to data analysisand completion. LIMS or LIS may automate sample logging at particularprocess steps by communicating with one or more applications or one ormore instruments. LIMS or LIS may log one or more additional informationrelated to the plate or sample, such as a scientist or an operator, areagent, an instrument, and a date or date and time of processing theplate or sample. A particular embodiment of LIMS or LIS may beintegrated into a fully automated system. A particular embodiment ofLIMS or LIS may require manual logging of one or more samples, one ormore plates, one or more operators, one or more reagents, one or moreinstruments, one or more processing date or times or where samples enteror exit specified processes.

In a particular embodiment, a system control may be the automatictitration to determine the optimal amount of binding element. In anotherembodiment, a system control is the use of “sample barcoding,” discussedbelow, to quickly and efficiently determine the optimal binding elementconcentration, thus reducing overall stain consumption.

Reagents

As described in detail throughout, the invention provides methods,compositions, devices and kits for high throughput flow cytometry. Insome embodiments, the invention provides methods, compositions, devicesand kits for phosphoflow cytometry and receptor function analysis. Assuch, there are numerous reagents commonly known in the art to beassociated with various embodiments of the invention. In particularembodiments, some reagents may be used at multiple process stepsthroughout sample preparation while others may be used only once. Thereare various reagents that may be added to the samples, plates, orchambers before or after addition of the cell sample, thus it is knownin the art that the order of reagent addition may vary. Reagents maycome in a variety of compositions, such as in solid (powder,lyophilized, etc.) or liquid form and may be used in the process inthese various forms as well. Reagents may also be stored and used atvarious temperatures throughout the process.

Modulators:

Modulators, also referred to as “stims,” include chemical and biologicalentities, and physical or environmental stimuli. Modulators can actextracellularly or intracellularly. Chemical and biological modulatorsmay include growth factors, cytokines, neurotransmitters, adhesionmolecules, hormones, small molecules, inorganic compounds,polynucleotides, antibodies, natural compounds, lectins, lactones,chemotherapeutic agents, biological response modifiers, carbohydrate,proteases and free radicals. Physical and environmental stimuli mayinclude electromagnetic or particulate radiation, redox potential andpH, the presence or absences of nutrients, changes in temperature,oxygen partial pressure, ion concentrations and oxidative stress.Modulators may be endogenous or exogenous and may produce differenteffects depending on the concentration and duration of exposure to thesingle cells or whether they are used in combination or sequentiallywith other modulators.

In particular embodiments, the modulators may include ligands for cellsurface receptors (for example the modulators include IL-2, EGF, GMCSF,etc). Examples of such receptor elements include compounds or eventsthat will activate hormone receptors, cytokine receptors, steroidreceptors, adhesion receptors and growth factor receptors, including,but not limited to, PDGF-R (platelet derived growth factor receptor),EGF-R (epidermal growth factor receptor), VEGF-R (vascular endothelialgrowth factor), uPAR (urokinase plasminogen activator receptor), ACHR(acetylcholine receptor), IgE-R (immunoglobulin E receptor), estrogenreceptor, thyroid hormone receptor, integrin receptors (β1, β2, β3, β4,β5, β6, α1, α2, α3, α4, α5, α6), MAC-1 (β2 and cd11b), αVβ33, opioidreceptors (mu and kappa), FC receptors, serotonin receptors (5-HT,5-HT6, 5-HT7), β-adrenergic receptors, insulin receptor, leptinreceptor, TNF receptor (tissue-necrosis factor), cytokine receptors(IL1-a, IL-b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10.IL-12, IL-15, IL-18, IL-21, CCR5, CCR7, CXCR4, CCR-1-10, CCL20), statinreceptors, FAS receptor, BAFF receptor, FLT3 receptor, GMCSF receptor,and fibronectin receptor. Specifically contemplated are modulators thatspecifically relate to any of the particular receptors noted above, suchas IL-2 for the IL-2 receptor, and VEGF-R for the VEGF receptor, forexample.

Commercially available modulators are: Phorbol 12-Myristate 13-Acetate(PMA); Thapsigargin, Lipopolysacharride (LPS), CD40L, SCF, IGF-1, IL-6,IL-10, Etoposide, IL-3, SDF-1a/CXCL12, HydroxyUrea, Z-VAD-FMK CaspaseInhibitor, G-CSF, Erythropoetin (EPO), SDF-1B/CXCL12, IL-27, M-CSF,GM-CSF, FLT-3 Ligand, VEGF, and TRAIL.

Other modulators may be selected from the group consisting of H₂O₂,siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR,Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodiumoxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV),Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole,Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate,Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin,Dephostatin, Okadaic Acid, NIPP-1,N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide,α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br,α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br,α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br,and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene,phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminium fluoride.

Inhibitors:

Inhibitors specifically prevent a specific type of cellular activity.Phosphatase inhibitors prevent the phosphatase enzyme from removing aphosphate group from a substrate, and thus preventing a specificcellular activity. Phosphatase inhibitors may be used to analyzepotential response to treatment or analyze non-response to treatment.Inhibitors may be used to correlate the expression or transporterproteins with their functionality. Inhibitors may be used to makecorrelations within subpopulations of cells gated both for phenotypicmarkers denoting stages of development along hematopoietic and lymphoidlineages, as well as reagents that recognize the transporter proteinsthemselves.

Inhibitors may include, but are not limited to, 3 mM hydrogen peroxide(H₂O₂), 3 mM H₂O₂+SCF and 3 mM H₂O₂+IFNα, PSC833, cyclosporine,probenecid, fumitremorgin C, Ko143, and reserpine.

Fixatives:

Particular embodiments of the system may utilize assay and screeningmethods, such as include “fixing ” Fixing may be performed to preserveor “freeze” a cell in a certain state, preferably so that an accuraterepresentation of the structure of the cell is maintained. In particularembodiments it may be desirable to fix a cell to maintain the cell'soriginal size and shape, to minimize loss of cellular materials, and/orto retain the reactivity and/or status of the cell's intracellularconstituents (for example, the cell's phosphorylation level).

In particular embodiments, cells may be fixed by any of a variety ofsuitable chemical and physical methods. In a particular embodiment,fixatives may be applied to the detection of both surface andintracellular antigens. In a particular embodiment, a fixing method maybe compatible with multi-well plate format assays. In a particularembodiment, one or more fixing agents may be added to cells contained inthe well of an assay plate. Cells may be incubated in the presence ofthe fixing agent at a certain temperature (for example at roomtemperature, i.e., between 18° C. and 25° C.) and for a certain periodof time (for example between 5 and 10 minutes). In a particularembodiment, fixation of cells in whole blood preferably wouldhypotonically lyse the red blood cells while simultaneously fixing andpreserving the white blood cells. An example whole blood fixative may befound here:http://www.bdbiosciences.com/external_files/pm/doc/tds/cellbio/live/web_enabled/558049.pdf.In particular embodiments, excess fixing agent may be removed aftercentrifugation by aspiration of the supernatant.

Particular embodiments of the system may rely on crosslinking and/orrapid dehydration agents, such as formaldehyde, paraformaldehyde,glutaraldehyde, acetic acid, methanol, ethanol, and acetone to fix thecells. In a particular embodiment, Ortho PERMEAFIX™, or PERMIFLOW™(INVIRION, INC.™ MI) may be used as a fixative. In a particularembodiment, a fixative may comprise 0.756%-0.85% formaldehyde, 25.4-30mM DNBS, 6.9-6.92% DMSO and 0.086-0.095% TWEEN™ 20 detergent, althoughmany variations may also be used.

In particular embodiments, wash buffers may be used to “fix” a cellafter stimulation with a modulator. Wash buffers are know in the art.See for example, U.S. Pat. No. 7,326,577 and U.S. Publication No.2006/0141549, which are hereby incorporated by reference in itsentireties.

Particular embodiments may utilize fixatives or fixing methods describedin the following references: Francis C. Connelly M C, Rapid single-stepmethod for flow cytometric detection of surface and intracellularantigens using whole blood, Cytometry. 1996 Sep. 1; 25(1):58-70;Jacobberger, J W, Flow Cytometric Analysis of Intracellular ProteinEpitopes. Immunophenotyping 2000; 361-409; S & Cheta N, PermeaFix: auseful tool to detect antigens and DNA in flow cytometry, Rom J InternMed. 1997 January-December; 35(1-4):133-5; Pizzolo G, et al. Detectionof membrane and intracellular antigens by flow cytometry following ORTHOPermeaFix fixation. Leukemia. 1994 April; 8(4):672-6; Pizzolo, G, etal., Detection of membrane and intracellular antigens by flow cytometryfollowing ORTHO PermeaFix fixation. Leukemia. 1994 April; 8(4):672-6);Metso, T, et al., Identification of intracellular markers in inducedsputum and bronchoalveolar lavage samples in patients with respiratorydisorders and healthy persons. Respir Med. 2002 November;96(11):918-26); U.S. Pat. No. 5,422,277 and U.S. Pat. No. 5,597,688.

Permeabilization:

Particular embodiments of the system may “permeabilize” cells once thecells have been fixed. Permeabilization may be performed to facilitateaccess to cellular cytoplasm or intracellular molecules, components orstructures of a cell. In particular, permeabilization may allow an agent(such as a phospho-selective antibody) to enter into a cell and reach aconcentration within the cell that is greater than that which wouldnormally penetrate into the cell in the absence of such permeabilizingtreatment. In particular embodiments, cells may be stored followingpermeabilization or combined with labeled binding elements and thenanalyzed. In a particular embodiment, cells may be permeabilized in thepresence of 90% methanol and incubated on ice for 30 minutes. Followingthis treatment, the assay plate may be stored at −20° C. for prior tobeing analyzed.

In particular embodiments, permeabilization of the cells may beperformed by any suitable method. Selection of an appropriatepermeabilizing agent and optimization of the incubation conditions andtime may easily be performed by one of ordinary skill in the art. Seefor example, C. A. Goncalves et al., Neurochem. Res. 2000, 25: 885-894.Suitable methods include, but are not limited to, exposure to adetergent (such as CHAPS, cholic acid, deoxycholic acid, digitonin,n-dodecyl-.beta.-D-maltoside, lauryl sulfate, glycodeoxycholic acid,n-lauroylsarcosine, saponin, and triton X-100) or to an organic alcohol(such as methanol and ethanol). Other permeabilizing methods comprisethe use of certain peptides or toxins that render membranes permeable.See, for example, O. Aguilera et al., FEBS Lett. 1999, 462: 273-277; andA. Bussing et al., Cytometry, 1999, 37: 133-139. Permeabilization mayalso be performed by addition of an organic alcohol to the cells.

Binding Element

In some embodiments of the invention, the activation level of anactivatable element is determined. Thus, the methods, compositions,devices and kits of the invention may be employed to examine and profilethe status of any activatable element in a cellular pathway, orcollections of such activatable elements. Activatable elements includeintracellular or extracellular biomolecules such as proteins, RNA, DNA,carbohydrates, metabolites, and the like. Activatable elements aredescribed in U.S. application Ser. No. 12/460,029 filed Jul. 10, 2009;Ser. No. 12/432,720 filed Apr. 29, 2009; and Ser. No. 12/229,476 filedAug. 21, 2008, incorporated herein by reference in their entirety.Single or multiple distinct pathways may be profiled (sequentially orsimultaneously), or subsets of activatable elements within a singlepathway or across multiple pathways may be examined (again, sequentiallyor simultaneously). As will be appreciated by those in the art, a widevariety of activation events can find use in the present invention. Ingeneral, the basic requirement is that the activation results in achange in the activatable protein that is detectable by some indication(termed an “activation state indicator”), preferably by altered bindingof a labeled binding element or by changes in detectable biologicalactivities (e.g., the activated state has an enzymatic activity whichcan be measured and compared to a lack of activity in the non-activatedstate). However, in other instances an activatable element getsactivated by increase expression. Thus, in those instances the increaseexpression of the activatable element will be measured whether or notthere is a moiety between two or more activation states of the cells.

In general, there are a variety of ways to detect the activation stateof a particular protein (i.e. activatible element). Particularembodiments of the system may utilize the state of the activatableprotein itself by directly assaying the activity or lack of activitywithin the signaling domains. In this embodiment, for example, the twoisoforms may be no activity (negative signal) versus kinase activity(measured using chromogenic substrates).

In some embodiment, the activation level of an activatable element isdetermined by contacting a cell with a binding element (“BE”) that isspecific for an activation state of the activatable element. The term“Binding element” includes any molecule, e.g., peptide, nucleic acid,small organic molecule which is capable of detecting an activation stateof an activatable element over another activation state of theactivatable element. Binding elements and labels for binding elementsare shown in U.S. Ser. No. /048,886; 61/048,920 and 61/048,657.

In some embodiments, the binding element is a peptide, polypeptide,oligopeptide or a protein. The peptide, polypeptide, oligopeptide orprotein may be made up of naturally occurring amino acids and peptidebonds, or synthetic peptidomimetic structures. Thus “amino acid”, or“peptide residue”, as used herein include both naturally occurring andsynthetic amino acids. For example, homo-phenylalanine, citrulline andnoreleucine are considered amino acids for the purposes of theinvention. The side chains may be in either the (R) or the (S)configuration. In some embodiments, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradation. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al.,Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which areexpressly incorporated by reference herein.

Particular embodiments of the system may utilize labeled bindingelements, or stains, or secondary stain reagents, which bindspecifically to one isoform of the protein. In some embodiments, theprotein BE may be an antibody. By “antibody” herein is meant a proteinconsisting of one or more polypeptides substantially encoded by all orpart of the recognized immunoglobulin genes. The recognizedimmunoglobulin genes, for example in humans, include the kappa (k),lambda (I), and heavy chain genetic loci, which together comprise themyriad variable region genes, and the constant region genes mu (u),delta (d), gamma (g), sigma (e), and alpha (a) which encode the IgM,IgD, IgG, IgE, and IgA isotypes respectively. Antibody herein is meantto include full length antibodies and antibody fragments, and may referto a natural antibody from any organism, an engineered antibody, or anantibody generated recombinantly for experimental, therapeutic, or otherpurposes as further defined below. The term “antibody” includes antibodyfragments, as are known in the art, such as Fab, Fab′, F(ab′)2, Fv,scFv, or other antigen-binding subsequences of antibodies, eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA technologies. Particularly preferred are fulllength antibodies that comprise Fc variants as described herein. Theterm “antibody” comprises monoclonal and polyclonal antibodies.Antibodies can be antagonists, agonists, neutralizing, inhibitory, orstimulatory.

Many antibodies, many of which are commercially available (for example,see Cell Signaling Technology's and Becton Dickinson's catalogues, thecontents which are incorporated herein by reference) have been producedwhich specifically bind to the phosphorylated isoform of a protein butdo not specifically bind to a non-phosphorylated isoform of a protein.Many such antibodies have been produced for the study of signaltransducing proteins that are reversibly phosphorylated. In particular,many such antibodies have been produced which specifically bind tophosphorylated, activated isoforms of protein kinases and are sometimesreferred to herein as kinase activation state antibodies or grammaticalequivalents thereof. Particularly preferred antibodies for use in thepresent invention include: phospho-AKT Ser473 monoclonal anti-4E2,phospho-p44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody,phospho-TYK2 (Tyr1054/1055) antibody, phospho-p38 MAP kinase(Thr180/Tyr182) monoclonal antibody 28B10, phospho-PKC-PAN substrateantibody, phospho-PKA-substrate, phospho-SAPK/JNK (Thr183/Tyr185) G9monoclonal antibody, phospho-tyrosine monoclonal antibody (P-tyr-100),p44/42 MAPK, p38 MAPK, JNK/SAPK, and phospho-AKT-Thr308.

In a particular embodiment, the protein BE may be an activationstate-specific antibody. Accordingly, the automation system may use themethods and compositions described to detect any particular elementisoform in a sample that is antigenically detectable and antigenicallydistinguishable from other isoforms of the activatible element. In aparticular embodiment, the system may use the activation state-specificantibodies to identify distinct signaling cascades of a subset orsubpopulation of complex cell populations, and the ordering of proteinactivation (e.g., kinase activation) in potential signaling hierarchies.

In a particular embodiment, the BE may be a stain, where stain may alsobe referred to as a detectable element, label or a tag. By label ismeant a molecule that can be directly (i.e., a primary label) orindirectly (i.e., a secondary label) detected. By way of example, andnot by way of limitation, a label may be visualized and/or measured orotherwise identified so that its presence or absence may be known. Acompound may be directly or indirectly conjugated to a label whichprovides a detectable signal, e.g. radioisotopes, fluorescers, enzymes,antibodies, particles such as magnetic particles, chemiluminescers,specific binding molecules, or molecules that may be detected by massspectroscopy, etc. Specific binding molecules include pairs, such asbiotin and streptavidin, digoxin and antidigoxin, etc. In particularexample embodiments, labels may be optical fluorescent and chromogenicdyes including labels, label enzymes radioisotopes, and quantum dots.

Kits

Particular embodiments of the high throughput flow cytometry system andmethod may utilize kits. Such kits may enable the detection ofactivatable elements by sensitive cellular assay methods, such as IHCand flow cytometry, which are suitable for the clinical detection,prognosis, and screening of cells and tissue from patients, such asleukemia patients, having a disease involving altered pathway signalingParticular embodiments may be compatible with clinical or researchapplications, a highly automated or less automated high throughput flowcytometry system, and a wide variety of instruments. Particularembodiments may be provided, marketed and/or promoted to healthproviders, including physicians, nurses, pharmacists, formularyofficials, and the like, or be marketed directly to the consumer.Particular embodiments may include reagents for sample preparation,consumable hardware, such as plates or other vessels for carrying thereagents or providing a reaction space or pipette tips or tubing, acomputer readable tangible medium storing a computer software program orsoftware files that contain kit details and processing methods requiredfor sample preparation, and information, such as package insertmaterials or literature references. Particular embodiments may bepackaged in any suitable manner, but typically with all elements in asingle container.

In a particular embodiment, reagents for sample preparation may includeone or more of the state-specific binding elements described herein,such as phospho-specific antibodies, and modulators, fixatives, buffers,therapeutic agents, and the like.

In a particular embodiment, one or more of the state-specific bindingelements may be phospho-specific antibodies specific for the proteins ofinterest. Examples of antibodies that may be used may be selected fromthe group: PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs, Rac,Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2,SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3,TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK,Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK,MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk,Btk, BLNK, LAT, ZAP70, Lck, Cbl, SLP-76, PLCγ□, PLCγ2, STAT1, STAT 3,STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2, Hsp90, Hsp70,Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb,Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, p14Arf, p27KIP, p21CIP,Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25, A/B/C, Abl, E2F, FADD, TRADD,TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1, Bcl-XL, Caspase 2, Caspase 3,Caspase 6, Caspase 7, Caspase 8, Caspase 9, IAPB, Smac, Fodrin, Actin,Src, Lyn, Fyn, Lck, NIK, Iβ□□1□□, PKCα, PKCβ, PKCγ□□P, βcatenin, CrkL,GSK3α, GSK3β, and FOXO.

In a particular embodiment, one or more of the phospho-specificantibodies specific for the proteins selected may be selected from thegroup consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLCγ2, Akt,RelA, p38, S6. In a particular embodiment, one or more of thephospho-specific antibodies specific for the proteins selected may beselected from the group consisting of Akt1, Akt2, Akt3, SAPK/JNK1,2,3,p38s, Erk1/2, Syk, ZAP70, Btk, BLNK, Lck, PLCγ, PLCγ 2, STAT1, STAT 3,STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, NF-κ□

In a particular embodiment, modulators may be included. In a particularembodiment, one or more modulators may be selected from the groupconsisting of H₂O₂, PMA, BAFF, April, SDF1 α, CD40L, IGF-1, Imiquimod,polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigardin and acombination thereof.

In a particular embodiment, the state-specific binding element may beconjugated to a solid support and to detectable groups directly orindirectly. In a particular embodiment, the reagents may also includeancillary agents such as buffering agents and stabilizing agents, e.g.,polysaccharides and the like. In a particular embodiment, the reagentsmay also include other members of the signal-producing system of whichsystem the detectable group is a member (e.g., enzyme substrates),agents for reducing background interference in a test, control reagents,apparatus for conducting a test, and the like. In a particularembodiment, one or more therapeutic agents may be included. The kit mayfurther comprise a software package for data analysis of thephysiological status, which may include reference profiles forcomparison with the test profile.

In a particular embodiment, a computer readable tangible medium,including discs, thumb drives, external hard drives, contains a computersoftware program or software files that contain kit details andprocessing methods required for sample preparation. The software mayinclude files for a specific reaction using specific reagents. In aparticular embodiment, the software may include directions for carryingout the process of reagent addition, incubation, fixing cells, and otherprocesses described herein. In a particular embodiment, the softwareprogram or software files may govern the positioning of the instrumentand the amount of time the reagents are allowed to react with the cellsamples. In a particular embodiment, a computer readable tangible mediumis compatible with an instrument, such as the instrument illustrated inFIG. 4.

Such kits may also comprise tools and reagents to isolate a biologicalspecimen from an individual. The kits of the invention may also comprisetools and reagent to isolate one or more components (e.g. cytokines)from the biological specimen.

Such kits may additionally comprise one or more therapeutic agents. Thekit may further comprise a software package for data analysis of thephysiological status, which may include reference profiles forcomparison with the test profile.

Particular embodiments may include information, such as scientificliterature references, package insert materials, clinical trial results,and/or summaries of these and the like, which indicate or establish theactivities and/or advantages of the composition, and/or which describedosing, administration, side effects, drug interactions, or otherinformation useful to the health care provider. Such information may bebased on the results of various studies, for example, studies usingexperimental animals involving in vivo models and studies based on humanclinical trials.

“Sample Barcoding”

Particular embodiments of the automation system may be adjusted toincrease the throughput through the use of a process referred to as“sample barcoding.” In particular embodiments, sample barcoding mayreduce or eliminate well to well variation, such as variations instaining and the resultant variation in fluroescence. In particularembodiments, sample barcoding may reduce overall antibody consumption.In particular embodiments, sample barcoding may allow multiple samplesthat have been processed differently to be combined into a single tubeor well prior to the staining step so that all samples may be analyzedtogether in a single run on the flow cytometer. See Fluorescent cellbarcoding inflow cytometry allows high-throughput drug screening andsignaling profiling, Krutzik, P. and Nolan G., Nature Methods, Vol. 3No. 5, Pgs. 361-365, May 2006; see also U.S. Patent Publication No.20080241820, which are hereby incorporated by reference in theirentirety.

In particular embodiments, sample barcoding may occur duringpermeabilization, where the samples are also labeled with thefluorophore barcodes. In particular embodiments, samples may be labeledwith different intensities of fluorophores by treatments with varyingconcentrations or combinations of the reactive forms of thefluorophores, which may result in each sample having a uniquefluorescence intensity signature. In particular embodiments, the use ofmultiple fluorophores may increase the number of different samples thatmay be barcoded, combined into a single well, and then analyzedtogether. In particular example embodiments, one fluorophore may allow4-6 samples to be analyzed together, two fluorophores may allow 16-36samples to be analyzed together, and three fluorophores may allow 64-216samples to be analyzed together. The greater the number of flurophoresused, the greater the number of samples that may be analyzed together.In particular embodiments, downstream analysis software may be used todeconvolute the data from the single well back into separate results foreach sample using the fluorescent barcodes.

In particular embodiments, sample barcoding may replace varioustechniques for optimizing the amount of binding element to add to aparticular sample. Binding element may be added to a single sample wellor tube containing both the unstim and the stim samples, which may thenbe analyzed together. In particular embodiments, false positives due toan overconcentration of antibodies may be quickly and accuratelyidentified in the unstim cells by allowing the data from the unstim andstim to be visualized together. In particular embodiments, samplebarcoding may indicate whether antibody concentrations are too weak ortoo strong. In particular embodiments, sample barcoding may permit theanalysis of small shifts in cell populations that may not otherwise benoticeable with other techniques.

In particular embodiments, sample barcoding may allow for distinctionbetween those cells that were dead at the beginning of processing andcells that died as a result of processing. A cell sample may be treatedwith one or more stains that are capable of distinguishing between liveand dead cells, such as an amine reactive dye. Examples of stainsinclude, but are not limited to, Aqua, Alexa (Alexa 750 among othertypes of Alexa dyes), and Sytox (available from Invitrogen oreBioscience). In a particular embodiment, two or more stains may be usedto detect the number of cells that lost viability after variousprocessing steps or at various time intervals. In particularembodiments, sample barcoding may allow gating between dead cells andlive cells to remove dead cells from further analysis, resulting incells being divided into three classifications.

Sample Analysis

Particular embodiments of the system may use a system or method for highthroughput sample analysis. In a particular embodiment, an instrumentmay be employed that provides for fast and automated sampling ofmicroplate wells. Such an instrument may be used to take the samples atthe point that they are ready to be analyzed by a flow cytometer orother detector. In a particular embodiment, an instrument may deliversamples to the flow cytometer for analysis.

An example instrument that is commercially available and may beintegrated into the system is the Hypercyt (Intellicyt, Albuquerque,N.M.). The HyperCyt® high-throughput flow cytometry approach is to fillthe sample uptake/transport line with a stream of discrete sampleparticle suspensions separated by an air gap, each sample suspensionbeing approximately 2 μl or larger if necessary. Particular embodimentsof the system may use 1, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more microlitersper sample. The entire sample stream is continuously delivered to theflow cytometer so that data from all the samples in a plate are acquiredand stored in a single data file. A high resolution time parameter isalso recorded during data acquisition. Temporal gaps in particledetection are created in the data stream by the passage of the airbubbles, allowing the individual particle suspensions to bedistinguished and separately evaluated when plotted in conjunction withthe time parameter. See U.S. Pat. No. 7,368,084;http://www.intellicyt.com, andhttp://www.intellicyt.com/download/SBS2007HyperCyt.pdf. In a particularembodiment, the Hypercyt instrument may be operably connected to theflow cytometer and provide a liquid connection for samples to beinserted into the cytometer at a high rate.

Data Analysis

Automated sample processing for flow cytometry analysis generates largeamounts of data in various formats. Particular embodiments of theautomation system contain a system for automated data analysis. In aparticular embodiment, automated gating of sample data may be used toautomate data analysis. U.S. Patent Application No. 61/079,579 is herebyfully incorporated by reference in its entirety. In a particularembodiment, automation system may utilize a method and system for dataextraction and visualization of multiparametric data. U.S. PatentApplication No. 61/079,537, hereby fully incorporated by reference inits entirety.

Applications

Applications of high throughput flow cytometry system and methods mayinclude, but are not limited to, probing various pathways by analyzingresponses to a host of stimuli, assessing the inhibition or enhancementof various signaling pathways by the additions of small compounds from asmall compound library, assessing expression level and functionality ofvarious drug transporters, or probing the signaling differences amongvarious cancer cell lines (ie, NCI 60 cell lines). Examples of drugtransporters include, but are not limited to, glycoprotein (MDR1),MDR-associated protein and breast cancer resistance protein. Inparticular embodiments, automation may be used to screen thesupernatants from hybridomas in order to pick the best antibodysecreting cell clone. In particular embodiments, automation may be usedto screen for a neutralizing or agonistic antibody to a growth factorreceptor by looking for the supernatant that best augments the signalingdownstream of the growth factor receptor. In particular embodiments,automation may be used to select the best cell clone secreting anyprotein that has a biological effect that could be measured by itsdownstream effect on signaling. In particular embodiments, highthroughput system may be used to determine the expression andfunctionality of cell surface proteins prior to treatment and inresponse to various treatments related to cancer therapy. Particularembodiments may analyze multiple characteristics of the cell in paralleland after contact with the therapy compound.

In particular embodiments, the system may be used for runningexperiments or portions of experiments including, but not limited to,apoptosis assays, cell stimulation assays, and kinetic timepoint assays.FIG. 5 illustrates an example cell sample preparation method forpreparing apoptosis, standard, and kinetic timepoint plates. Thefollowing processing steps are illustrated in FIG. 5:

1. Use laboratory information system (LIS) or other automation softwareto initiate Liquid Handler Program.

2. System requests appropriate stock stim/modulator to be loaded.

3. System requests appropriate diluents, tips, tubes and plates forloading.

4. System loads and reads barcodes for samples, stims/modulators, tips,tubes, plates, etc.

5. System performs Stim/Modulator dilutions to create working solutions(WS) in tubes.

6. System aliquots WS into Master Stim Plates for 96 Head Stamping (All96 wells and/or Row/Column Format).

7. System aliquots samples and controls to designated layouts inApoptosis, Standard and Kinetic Timepoint Plates.

8. System places standard and kinetic timepoint plates on heatedshakers.

-   -   a. Temperature is controlled to ±0.5° C.    -   b. Setpoint may be adjusted and controlled between 30° C. and        50° C.    -   c. Shaking/Vortex may be adjusted and controlled between 200 rpm        to 1500 rpm with 3 mm amplitude

9. System logs all barcodes, source positions, volume pipetted anddestination positions.

FIG. 6 illustrates an example apoptosis assay method. The followingprocessing steps are further illustrated in FIG. 6:

1. Apoptosis working solutions are pipetted according to designatedformat and number of samples to achieve final stim/modulatorconcentrations in each well. This step may require scheduling softwareto maintain acceptable time requirements.

2. Plate is moved to 37° C. Temperature Controlled CO2 Incubator forspecified incubation time.

3. Plate is brought out of CO2 Incubator and Fix/Lyse Reagent added withtemperature and time control around fixation.

4. Plate is centrifuged, aspirated and cells dispersed by vortex.

5. Permeablization Reagent is added to plate and stored for designatedtime at −80° C.

6. Plate is centrifuged, aspirated, cells dispersed by vortex and washbuffer added.

7. Plate is centrifuged, aspirated and cells dispersed by vortex.

8. System loads Apoptosis Stain Cocktails, pipettes to designated wells,vortexes to mix, and incubates under time and temperature control (4° C.or 25° C.).

9. Wash buffer is added, the plate is centrifuged, aspirated, cellsdispersed by vortex and wash buffer added.

10. Plate incubates for set time with wash buffer.

11. Plate is centrifuged, aspirated and cells dispersed by vortex.

12. Secondary stain reagents are loaded and pipetted according to layoutand number of samples.

13. Plate is incubated for set time, wash buffer added, the plate iscentrifuged, aspirated, cells dispersed and resupsended to a finalvolume and cell concentration.

14. Samples are transferred from Deep Well to shallow well plate foracquisition (or to tube based carousel for acquisition) on cytometer.

FIG. 7 illustrates an example standard stimulation assay method. FIG. 8illustrates an example kinetic timepoint assay method. Processing stepsfor a stimulation assay method and a kinetic timepoint assay method maybe similar, but may differ at the step of adding the standardizedmodulator working solutions or the kinetic timepoint modular workingsolutions respectively. The following process steps illustrate examplestandard stimulation and kinetic timepoint assay processing:

1. Standard Stim Plate or Kinetic Timepoint Stim Plate is moved to deepwell heater/shaker and gently vortexed for predetermined time to obtaindesired stimulation temperature.

2. Standardized Stim/Modulator working solutions and Kinetic TimepointStim/Modulator working solutions are pipetted into plate:

-   -   a. For Standarized Stim/Modulator (as illustrated in FIG. 7):        working solutions are already in desired Master Plate layout.        -   i. Working solutions are pipetted by 96 Head to all wells at            once.        -   ii. Plate is maintained under Temperature Control on the            heater/shaker for specified stim incubation time.    -   b. For Kinetic Timepoint Stim/Modulator (as illustrated in FIG.        8): working solutions are already in desired Master Plate        layout.        -   i. At t=0, the first set of wells (rows or columns) have            stim/modulator added, timer is started and the plate is            vortexed briefly to assure uniform mixing and maintained at            under Temperature Control on the heater/shaker for specified            stim incubation time.        -   ii. At an appropriate time point, the next series of            stim/modulators are added, vortexed briefly to assure            uniform mixing and maintained at under temperature control            on the heater/shaker for specified stim incubation time. 1,            2, 3, or more iterations of this step may occur.

3. This step may require scheduling software to maintain acceptable timerequirements. At designed Stim Time (t_(stim)), Fix/Lyse Reagent isadded while maintaining temperature and time control around fixation.

4. Plate is centrifuged, aspirated and cells dispersed by vortex.

5. Perm Reagent is added to plate and stored for designated time at −80°C.

6. Plate is centrifuged, aspirated, cells dispersed by vortex and washbuffer added.

7. Plate is centrifuged, aspirated and cells dispersed by vortex.

8. System loads Standard Stain Cocktails, pipets to designateddestination according to layout and number of samples, vortex to mix andincubates under time and temperature control (4° C. or 25° C.).

9. Wash buffer is added, the plate is centrifuged, aspirated, cellsdispersed by vortex and wash buffer added.

10. Plate incubates for set time with wash buffer.

11. Plate is centrifuged, aspirated and cells dispersed by vortex.

12. Secondary Stain Reagents are loaded and pipetted according to layoutand number of samples.

13. Plate is incubated for set time, wash buffer added, the plate iscentrifuged, aspirated, cells dispersed and resupsended to a finalvolume and cell concentration.

14. Samples are transferred from Deep Well to shallow well plate foracquisition (or to tube based carousel for acquisition) on cytometer.

FIG. 9 illustrates an example high throughput multi-drug resistantfunctional efflux assay method that may be used to determine expressionand functionality of specific cell surface proteins prior to and inresponse to a given therapy. The following steps illustrate the highthroughput multi-drug resistant functional efflux assay method ofprocessing:

1. Use laboratory information system (LIS) or other automation softwareto initiate Liquid Handler Program.

2. Cell sample plates are prepared according to cell type by eitherthawing and resuspending, resuspending, or isolating and resuspending.RPMI 10% FCS buffer and/or RPMI 10% FBS buffer may be added to eachsample as necessary.

3. Inhibitor is prepared to a specific concentration and stored at 37°C. until use.

4. Appropriate amount of inhibitor is added to sample plate, mixed, andincubated for 15 minutes at 37° C.

5. Dye solution is prepared from concentrated dye and DPBS/0.5% BSA.

6. Plates are removed from incubator 30 seconds prior to adding dyesolution, and then appropriate amount of dye solution is added to sampleplate, mixed, and incubated for 45 minutes at 37° C.

7. Plate is centrifuged, aspirated and cells dispersed by vortex.

8. Efflux buffer is prepared using RPMI 10% FCS.

9. Appropriate amount of efflux buffer is added to sample plate,incubated at 37° C. for 20 minutes.

10. Plate is centrifuged, aspirated and cells dispersed by vortex.

11. IgG staining solution is prepared and added to sample plate, mixed,vortexed, and incubated on ice (or equivalent temperature) for 5minutes.

12. Plate is centrifuged, aspirated and cells dispersed by vortex.

13. Specific antibody and isotype staining mixtures are prepared andadded to sample plate, mixed, vortexed, and incubated on ice (orequivalent temperature) in the dark for 30 minutes.

14. Plate is centrifuged, aspirated and cells dispersed by vortex.

15. Wash buffer is added, plate is centrifuged, aspirated, cellsdispersed by vortex.

16. After final wash/aspiration, cells are dispersed and resupsended toa final volume and cell concentration.

17. Samples are transferred from Deep Well to shallow well plate foracquisition (or to tube based carousel for acquisition) on cytometer.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. Many other schemes fordistributing functions among the various functional elements of theillustrated embodiment are possible. The functions of any element may becarried out in various ways in alternative embodiments. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention. Therefore,the present disclosure encompasses all changes, substitutions,variations, alterations, and modifications to the example embodimentsherein that a person having ordinary skill in the art would comprehend.Similarly, where appropriate, the appended claims encompass all changes,substitutions, variations, alterations, and modifications to the exampleembodiments herein that a person having ordinary skill in the art wouldcomprehend. Thus, it is intended that the following claims define thescope of the invention and that methods and structures within the scopeof these claims and their equivalents be covered thereby.

1.-58. (canceled)
 59. A method for automated or semi-automatedprocessing of samples and multiparametric analysis of the processedsamples in a cytometer, comprising: a. providing a system for processingsamples to produce processed samples prior to analyzing the processedsamples in the cytometer, comprising:
 1. at least one microplate andmicroplate holder;
 2. a first reagent container containing a firstdetectable binding element that is specific for an activation state of afirst activatable element, a second reagent container containing asecond detectable binding element that is specific for a first cellsurface marker, wherein the first and second detectable binding elementsare differentially detectable by the cytometer, a third and fourthreagent containers containing a first and second modulator,respectively, wherein the detectable binding elements and the modulatorsare in liquid solution or suspension in their respective reagentcontainers;
 3. a dispensing head configured to withdraw liquid from oneof said reagent or sample containers and dispense it to a well in saidmicroplate, and/or to withdraw liquid from a first well in themicroplate and dispense it to a second well in the same or differentmicroplate;
 4. a microplate handling apparatus for moving saidmicroplate, wherein: i. said microplate handling apparatus is configuredto position said microplate near said dispensing head so that saiddispensing head can dispense liquid from a reagent container into a wellin said microplate and/or withdraw liquid from a well in said microplateduring the production of a processed sample or to position saidmicroplate so that it can be manually moved to be near said dispensinghead; and ii. said microplate handling apparatus is configured to eitherrelocate said microplate to said cytometer for transfer of the processedsample to, and analysis of the processed sample by, the cytometer, or toposition said microplate so that it may be relocated manually to saidcytometer for transfer of the processed sample to, and analysis of theprocessed sample by the cytometer; b. providing a system for analysisand management of the processed samples in step a, comprising:
 1. saidcytometer, wherein the cytometer is configured to differentially detecta plurality of differentially detectable binding elements bound toactivatable elements and cell surface markers in single cells present insaid samples; and
 2. a computer system including i. a processor, ii. alaboratory information management system (LIMS) configured to track andrecord an experiment, inventory management and experimental design andto automate sample tracking from wherever the sample is initially inputinto the automation system through to data analysis and completion, andiii. a memory unit configured to control, and operably connected to,said microplate holder, said tracking mechanism, said microplatehandling apparatus, said dispensing head, and said cytometer, whereinthe memory unit and the LIMS are configured to communicate with thecytometer by instrument control and image processing applicationsoftware to inform the cytometer as to plate layout, including thepresence or absence of the first or second differentially detectablebinding elements, or combination thereof, for each well in the plate,wherein there are potentially at least 6 differentially detectablebinding elements and each well of the plate may contain one or moredifferent binding elements compared to other wells, as well as thepresence or absence of one or more modulators, with correspondingmodulation time, so as to direct proper analysis of each of the samplesin each well of the plate by the cytometer, in a manner compatible withthe cytometer software, wherein the computer system is configured toconsolidate elements of data or metadata to a single file; c.withdrawing and dispensing liquids from said reagent containers into oneor more wells of the at least one microplate to perform a protocol,wherein said protocol comprises:
 1. preparing a master modulator platein an automated process wherein the first modulator is removed from thethird reagent container and dispensed into a first well in themicroplate and the second modulator is removed from the fourth reagentcontainer and dispensed into a second well in the microplate; 2.providing a second microplate comprising a first well and a second well,each of which contains a biological sample comprising cells, anddispensing the first modulator from the first well in the firstmicroplate to the first well in the second microplate and the secondmodulator from the second well in the first microplate to the secondwell in the second microplate, so that the modulators and the cells arein contact, wherein said dispensing may be manual or automated or acombination of manual and automated; and
 3. determining an activationlevel of at least said first activatable element in single cells in saidone or more samples, wherein said first activatable element is anelement in a pathway that is modulated by the first modulator or thesecond modulator, by said cytometer, wherein said activation level isdetermined by a process comprising: contacting said at least onebiological sample with the first detectable binding element which isspecific to an activation state of said first activatable element,wherein said contacting comprises: withdrawing said first detectablebinding element from said first reagent container and dispensing saidfirst detectable binding element to the well in the microplatecontaining said sample such that said first detectable binding elementcontacts the cells in said sample and binds to the first activatableelement, if present, and detecting said bound first detectable bindingelement in said cells on a single cell basis in the cytometer; and 4.determining a level of at least said first cell surface marker in saidone or more samples on a single cell basis by said cytometer, whereinsaid first cell surface marker level is determined by a processcomprising: contacting said at least one biological sample with thesecond detectable binding element which is specific to the first cellsurface marker, wherein said contacting comprises: withdrawing saidsecond detectable binding element from said second reagent container anddispensing said second detectable binding element to the well in themicroplate containing said sample such that said second detectablebinding element contacts the cells in said sample and binds to saidfirst cell surface marker, if present, and detecting said bound seconddetectable binding element in said cells on a single cell basis in thecytometer.
 60. The method of claim 59 wherein the automated process ofc.1 comprises (a) initiating a liquid handler program using automatedsoftware; (b) system requesting an appropriate stock modulator to beloaded into automated system; and (c) automated system automaticallyperforms modulator dilutions to create working solutions for the firstmodulator in the third reagent container and the second modulator in thefourth reagent container, according to liquid handler program.
 61. Themethod of claim 59 wherein said samples comprise intact viable cells andwherein said pre-processing comprises contacting said intact viablecells with the modulator for a predetermined time, and wherein thecontacting of step c.2 further comprises contacting the cells and themodulator in such a way that the sample remains viable during thecontacting for the predetermined time.
 62. The method of claim 61wherein the computer system is configured to control the contacting ofthe cells with the modulator for the predetermined time.
 63. The methodof claim 62 wherein the computer system controls the contacting of thecells with modulator for the predetermined time by controlling a processcomprising fixing of the sample after treatment with the modulator. 64.The method of claim 63 wherein said process comprising fixing of thesample after treatment with the modulator comprises withdrawing thefixative from a reagent container or containers and dispensing thefixative to the wells of the at least one microplate containing thesample, or withdrawing said sample from a sample container anddispensing the sample to wells of the at least one microplate containingthe fixative, such that the fixative contacts the cells of the sample.65. The method of claim 64 wherein the computer system controls thecontacting of the cells with the fixative for a predetermined time. 66.The method of claim 59 wherein the system further comprises at least oneof a fifth reagent container containing a third detectable bindingelement specific to an activation state of a second activatable element,a sixth reagent container containing a fourth detectable binding elementspecific to an activation state of a third activatable element, or aseventh reagent container containing a fifth detectable binding elementspecific to an activation state of a fourth activatable element, whereinthe third, fourth, and fifth detectable binding elements aredifferentially detectable from each other and from the first and secondbinding elements, and wherein each detectable binding element iscontacted with cells in sample in at least one well of the at least onemicroplate and detected by the cytometer.
 67. The method of claim 66wherein the system comprises at least two of the fifth, sixth, andseventh reagent containers and their respective detectable bindingelements.
 68. The method of claim 67 wherein the system furthercomprises a reagent container containing a third modulator and wherein,in step c.1, the third modulator is removed from its reagent containerand dispensed into a third well of the master modulator plate.
 69. Themethod of claim 68 wherein the system further comprises a reagentcontainer containing a fourth modulator and wherein, in step c.1, thefourth modulator is removed from its reagent container and dispensedinto a fourth well of the master modulator plate.
 70. The method ofclaim 69 wherein the system further comprises a reagent containercontaining a fifth modulator and wherein, in step c.1, the fifthmodulator is removed from its reagent container and dispensed into afifth well of the master modulator plate.
 71. The method of claim 70wherein the first, second, third, fourth, and fifth modulators aredifferent from each other and the first, second, third, fourth, andfifth wells are different from each other.
 72. The method of claim 59wherein the cytometer is a flow cytometer.
 73. The method of claim 59wherein the cytometer is a mass cytometer.
 74. The method of claim 59wherein the single file is not duplicated.
 75. A method for automated orsemi-automated processing of samples and analysis of the processedsamples by a cytometer, comprising: a. providing a system for processingsaid samples prior to their analysis in a cytometer, comprising:
 1. atleast one or more microplates and microplate holder;
 2. a first reagentcontainer containing a first detectable binding element that is specificfor an activation state of a first activatable element, a second reagentcontainer containing a second detectable binding element that isspecific for a first cell surface marker, wherein the first and seconddetectable binding elements are differentially detectable, and either athird reagent container containing a first modulator or a samplecontainer containing a biological sample comprising cells, or both,wherein the modulator, the detectable binding elements, and thebiological sample comprising cells are in liquid solution or suspensionin their respective reagent containers;
 3. a dispensing head configuredto withdraw liquid from one of said reagent or sample containers anddispense it to a well in said microplate and/or to withdraw liquid froma first well in the microplate and dispense it to a second well in thesame or different microplate;
 4. a microplate handling apparatus formoving said microplate, wherein; i. said microplate handling apparatusis configured to position said microplate near said dispensing head sothat said dispensing head can dispense liquid from a reagent containerinto a well in said microplate and/or withdraw liquid from a well insaid microplate during the production of a processed sample or toposition said microplate so that it can be manually moved to be nearsaid dispensing head; and ii. said microplate handling apparatus isconfigured to either relocate said microplate to said cytometer fortransfer of the processed sample to, and analysis of the processedsample by, the cytometer, or to position said microplate so that it maybe relocated manually to said cytometer for transfer of the processedsample to, and analysis of the processed sample by the cytometer; b.providing a system for, analysis and management of the processed samplesin step a, comprising:
 1. said cytometer, wherein the cytometer isconfigured to differentially detect a plurality of differentiallydetectable binding elements bound to activatable elements and cellsurface markers in single cells present in said samples;
 2. a computersystem, wherein said computer system comprises i. a processor, ii. aLIMS configured to track and record an experiment design and to automatesample tracking from wherever the sample is initially input into saidautomation process and analysis through to data analysis and completion,and iii. a memory unit configured to control, and operably connected to,said microplate holder, said barcode reader, said microplate handlingapparatus, the timing of said microplate handling steps, said dispensinghead, and said cytometer, wherein the memory unit and the LIMS areconfigured to communicate with the cytometer by instrument control andimage processing application software to inform the cytometer as toplate layout, including the presence or absence of the first or seconddifferentially detectable binding elements, or combination thereof, foreach well in the plate, wherein there are potentially at least 6differentially detectable binding elements and each well of the platemay contain one or more different binding elements compared to otherwells, as well as the presence or absence of one or more modulators,with corresponding modulation time, so as to direct proper analysis ofeach of the samples in each well of the plate by the cytometer, in amanner compatible with the cytometer software, wherein the computersystem is configured to consolidate elements of data or metadata to asingle file; c. withdrawing and dispensing liquids from said reagentcontainers into one or more wells of the at least one microplate toperform a protocol, wherein said protocol comprises:
 1. either: i.providing the at least one microplate, one or more of whose wellscontains biological sample comprising cells, and dispensing modulatorfrom said third reagent container into the wells so that the modulatorand the cells are in contact; or ii. providing the at least onemicroplate, one or more of whose wells contains a modulator anddispensing at least one biological sample comprising cells from saidsample container into the wells so that the modulator and the cells arein contact; or iii. providing the at least one microplate and dispensingat least one biological sample from the sample container into one ormore wells of the microplate and dispensing modulator from the thirdreagent container into one or more of the wells of the microplate, sothat the modulator and the cells are in contact; and
 2. contacting saidsamples with a fixative, wherein said contacting is performed byautomation at a time determined by the LIMs, and a permeablizationreagent;
 3. determining an activation level of at least said firstactivatable element in single cells in said one or more samples whereinsaid first activatable element is an element in a pathway that ismodulated by the modulator, by said cytometer, wherein said activationlevel is determined by a process comprising: contacting said at leastone biological sample with the first detectable binding element which isspecific to an activation state of said first activatable element,wherein said contacting comprises: withdrawing said first detectablebinding element from said first reagent container and dispensing saidfirst detectable binding element to the well in the microplatecontaining said sample such that said first detectable binding elementcontacts the cells in said sample and binds to the first activatableelement, if present, and detecting said bound first detectable bindingelement in said cells on a single cell basis in the cytometer; 4.determining a level of at least said first cell surface marker in saidone or more samples on a single cell basis by said cytometer, whereinsaid first cell surface marker level is determined by a processcomprising: contacting said at least one biological sample with thesecond detectable binding element which is specific to the first cellsurface marker, wherein said contacting comprises: withdrawing saidsecond detectable binding element from said second reagent container anddispensing said second detectable binding element to the well in themicroplate containing said sample such that said second detectablebinding element contacts the cells in said sample and binds to saidfirst cell surface marker, if present, and detecting said bound seconddetectable binding element in said cells on a single cell basis in thecytometer; d. wherein the LIMS software is configured to directexperimental design, plate layout, sample tracking, and inventorymanagement and one or more of the following purposes from the groupconsisting of instrument alignment; correct connections; motoroperations; timing for sample handling; and data analysis.
 76. Themethod of claim 75, wherein the steps in the protocol are conducted inislands of automation.
 77. The method of claim 75, wherein one or morecomponents of the system are in a thermo regulating component.
 78. Themethod of claim 75, wherein the microplate handling apparatus comprisesa robotic arm, and further comprising moving the at least one microplateusing the robotic arm.
 79. A method for processing of samples andanalysis of the processed samples in a cytometer, comprising: (i)preparing a master modulator microplate, wherein the master modulatorplate comprises at least a first well or tube containing a firstmodulator at a first concentration and a second well or tube containinga second modulator at a second concentration, each of which contains oneof a plurality of modulators, each modulator being present at apredetermined concentration for that modulator, comprising (a)initiating a liquid handler program using automated software; (b)loading stock modulator for each well into automated system according toa system request, wherein the loading may be automated or manual; (c)automatically performing modulator dilutions by the automated system tocreate working solutions in working solution containers according toliquid handler program; (d) automatically aliquoting working solutionsinto master modulator plate by automated system, thus preparing a mastermodulator plate according to liquid handler program; (ii) providing asample microplate, comprising a first well and a second well, each ofwhich contains a biological sample comprising cells, and dispensing thefirst modulator from the first well in the master modulator microplateto the first well in the sample microplate and the second modulator fromthe second well in the master modulator microplate to the second well inthe sample microplate, so that the modulators and the cells are incontact, wherein said dispensing may be manual or automated or acombination of manual and automated; (iii) adding fixative to the firstwell of the sample microplate at a first time after modulator and cellscome in contact and to the second well of the sample microplate at asecond time after modulator and cells come in contact; and (iv)analyzing single cells from the first well in a cytometer for thepresence of a first activatable element and analyzing cells from thesecond well in the cytometer for the presence of a second activatableelement.